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Structural and functional insights into the unique CBS-CP12 fusion protein family in cyanobacteria.
Hackenberg C, Hakanpaa J, Cai F, Antonyuk S, Eigner C, Meissner S, Laitaoja M, Janis J, Kerfeld CA, Dittmann E, Lamzin VS
Cyanobacteria are important photosynthetic organisms inhabiting a range of dynamic environments. This phylum is distinctive among photosynthetic organisms in containing genes encoding uncharacterized cystathionine beta-synthase (CBS)-chloroplast protein (CP12) fusion proteins. These consist of two domains, each recognized as stand-alone photosynthetic regulators with different functions described in cyanobacteria (CP12) and plants (CP12 and CBSX). Here we show that CBS-CP12 fusion proteins are encoded in distinct gene neighborhoods, several unrelated to photosynthesis. Most frequently, CBS-CP12 genes are in a gene cluster with thioredoxin A (TrxA), which is prevalent in bloom-forming, marine symbiotic, and benthic mat cyanobacteria. Focusing on a CBS-CP12 from Microcystis aeruginosa PCC 7806 encoded in a gene cluster with TrxA, we reveal that the domain fusion led to the formation of a hexameric protein. We show that the CP12 domain is essential for hexamerization and contains an ordered, previously structurally uncharacterized N-terminal region. We provide evidence that CBS-CP12, while combining properties of both regulatory domains, behaves different from CP12 and plant CBSX. It does not form a ternary complex with phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase. Instead, CBS-CP12 decreases the activity of PRK in an AMP-dependent manner. We propose that the novel domain architecture and oligomeric state of CBS-CP12 expand its regulatory function beyond those of CP12 in cyanobacteria.
Proc Natl Acad Sci U S A. 2018 Jun 18. pii: 1806668115. doi: 10.1073/pnas.1806668115.   PUBMED

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase.
Petrova OA, Mantsyzov AB, Rodina EV, Efimov SV, Hackenberg C, Hakanpaa J, Klochkov VV, Lebedev AA, Chugunova AA, Malyavko AN, Zatsepin TS, Mishin AV, Zvereva MI, Lamzin VS, Dontsova OA, Polshakov VI
The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.
Nucleic Acids Res. 2018 Feb 16;46(3):1525-1540. doi: 10.1093/nar/gkx1275.   PUBMED


A distance geometry-based description and validation of protein main-chain conformation.
Pereira J, Lamzin VS
Understanding the protein main-chain conformational space forms the basis for the modelling of protein structures and for the validation of models derived from structural biology techniques. Presented here is a novel idea for a three-dimensional distance geometry-based metric to account for the fine details of protein backbone conformations. The metrics are computed for dipeptide units, defined as blocks of C(alpha)i-1-O i-1-C(alpha)i -O i -C(alpha)i+1 atoms, by obtaining the eigenvalues of their Euclidean distance matrices. These were computed for approximately 1.3 million dipeptide units collected from nonredundant good-quality structures in the Protein Data Bank and subjected to principal component analysis. The resulting new Euclidean orthogonal three-dimensional space (DipSpace) allows a probabilistic description of protein backbone geometry. The three axes of the DipSpace describe the local extension of the dipeptide unit structure, its twist and its bend. By using a higher-dimensional metric, the method is efficient for the identification of C(alpha) atoms in an unlikely or unusual geometrical environment, and its use for both local and overall validation of protein models is demonstrated. It is also shown, for the example of trypsin proteases, that the detection of unusual conformations that are conserved among the structures of this protein family may indicate geometrically strained residues of potentially functional importance.
IUCrJ. 2017 Aug 8;4(Pt 5):657-670. doi: 10.1107/S2052252517008466. eCollection 2017 Sep 1.   PUBMED

Estimation of the protein-ligand interaction energy for model building and validation.
Beshnova DA, Pereira J, Lamzin VS
Macromolecular X-ray crystallography is one of the main experimental techniques to visualize protein-ligand interactions. The high complexity of the ligand universe, however, has delayed the development of efficient methods for the automated identification, fitting and validation of ligands in their electron-density clusters. The identification and fitting are primarily based on the density itself and do not take into account the protein environment, which is a step that is only taken during the validation of the proposed binding mode. Here, a new approach, based on the estimation of the major energetic terms of protein-ligand interaction, is introduced for the automated identification of crystallographic ligands in the indicated binding site with ARP/wARP. The applicability of the method to the validation of protein-ligand models from the Protein Data Bank is demonstrated by the detection of models that are `questionable' and the pinpointing of unfavourable interatomic contacts.
Acta Crystallogr D Struct Biol. 2017 Mar 1;73(Pt 3):195-202. doi: 10.1107/S2059798317003400. Epub 2017 Mar 6.   PUBMED

Synthesis, SAR and molecular docking study of novel non-beta-lactam inhibitors of TEM type beta-lactamase.
Antipin RL, Beshnova DA, Petrov RA, Shiryaeva AS, Andreeva IP, Grigorenko VG, Rubtsova MY, Majouga AG, Lamzin VS, Egorov AM
The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type beta-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.
Bioorg Med Chem Lett. 2017 Apr 1;27(7):1588-1592. doi: 10.1016/j.bmcl.2017.02.025. Epub 2017 Feb 16.   PUBMED


Novel non-beta-lactam inhibitor of beta-lactamase TEM-171 based on acylated phenoxyaniline.
Grigorenko VG, Andreeva IP, Rubtsova MY, Deygen IM, Antipin RL, Majouga AG, Egorov AM, Beshnova DA, Kallio J, Hackenberg C, Lamzin VS
The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-beta-lactam beta-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial beta-lactamase TEM-171, with a Ki of 88 muM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A beta-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A beta-lactamases. We also hypothesise that the presented route for finding non-beta-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.
Biochimie. 2017 Jan;132:45-53. doi: 10.1016/j.biochi.2016.10.011. Epub 2016 Oct 19.   PUBMED


Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging.
Giewekemeyer K, Hackenberg C, Aquila A, Wilke RN, Groves MR, Jordanova R, Lamzin VS, Borchers G, Saksl K, Zozulya AV, Sprung M, Mancuso AP
The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample's electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample--a budding yeast cell (Saccharomyces cerevisiae)--using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution.
Biophys J. 2015 Nov 3;109(9):1986-95. doi: 10.1016/j.bpj.2015.08.047.   PUBMED


Automated identification of crystallographic ligands using sparse-density representations.
Carolan CG, Lamzin VS
A novel procedure for the automatic identification of ligands in macromolecular crystallographic electron-density maps is introduced. It is based on the sparse parameterization of density clusters and the matching of the pseudo-atomic grids thus created to conformationally variant ligands using mathematical descriptors of molecular shape, size and topology. In large-scale tests on experimental data derived from the Protein Data Bank, the procedure could quickly identify the deposited ligand within the top-ranked compounds from a database of candidates. This indicates the suitability of the method for the identification of binding entities in fragment-based drug screening and in model completion in macromolecular structure determination.
Acta Crystallogr D Biol Crystallogr. 2014 Jul;70(Pt 7):1844-53. doi: 10.1107/S1399004714008578. Epub 2014 Jun 29.   PUBMED

Identification of additional telomerase component of the yeast H. polymorpha is a step towards understanding the complex at the atomic level.
Petrova OA, Smekalova EM, Zvereva ME, Lamzin V, Dontsova OA
no abstract available
Dokl Biochem Biophys. 2014 Mar;455(1):59-64. doi: 10.1134/S1607672914020057. Epub 2014 May 3.   PUBMED

Role of kappa-->lambda light-chain constant-domain switch in the structure and functionality of A17 reactibody.
Ponomarenko N, Chatziefthimiou SD, Kurkova I, Mokrushina Y, Mokrushina Y, Stepanova A, Smirnov I, Avakyan M, Bobik T, Mamedov A, Mitkevich V, Belogurov A Jr, Fedorova OS, Dubina M, Golovin A, Lamzin V, Friboulet A, Makarov AA, Wilmanns M, Gabibov A
The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant kappa light chain by the lambda chain (A17lambda), and the X-ray structure of A17lambda has been determined at 1.95 A resolution. It was found that compared with A17kappa the active centre of A17lambda is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126 degrees compared with 138 degrees in A17kappa. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17kappa and A17lambda variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.
Acta Crystallogr D Biol Crystallogr. 2014 Mar;70(Pt 3):708-19. doi: 10.1107/S1399004713032446. Epub 2014 Feb 15.   PUBMED


Visual automated macromolecular model building.
Langer GG, Hazledine S, Wiegels T, Carolan C, Lamzin VS
Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user.
Acta Crystallogr D Biol Crystallogr. 2013 Apr;69(Pt 4):635-41. doi: 10.1107/S0907444913000565. Epub 2013 Mar 14.   PUBMED


Internal structure of an intact Convallaria majalis pollen grain observed with X-ray Fresnel coherent diffractive imaging.
Mancuso AP, Groves MR, Polozhentsev OE, Williams GJ, McNulty I, Antony C, Santarella-Mellwig R, Soldatov AV, Lamzin V, Peele AG, Nugent KA, Vartanyants IA
We have applied Fresnel Coherent Diffractive Imaging (FCDI) to image an intact pollen grain from Convallaria majalis. This approach allows us to resolve internal structures without the requirement to chemically treat or slice the sample into thin sections. Coherent X-ray diffraction data from this pollen grain-composed of a hologram and higher resolution scattering information-was collected at a photon energy of 1820 eV and reconstructed using an iterative algorithm. A comparison with images recorded using transmission electron microscopy demonstrates that, while the resolution of these images is limited by the available flux and mechanical stability, we observed structures internal to the pollen grain-the intine/exine separations and pore dimensions-finer than 60 nm. The potential of this technique for further biological imaging applications is discussed.
Opt Express. 2012 Nov 19;20(24):26778-85.   PUBMED

Purification, biochemical characterization, and structure of recombinant endo-1,4-beta-xylanase XylE.
Fedorova TV, Chulkin AM, Vavilova EA, Maisuradze IG, Trofimov AA, Zorov IN, Khotchenkov VP, Polyakov KM, Benevolensky SV, Koroleva OV, Lamzin VS
The gene xylE encoding endo-1,4-beta-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding beta-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70 degrees C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period tau(1/2) of 7 h at 60 degrees C. The kinetic parameters were 0.52 mg/ml (K(m)) and 75 micromol/min per mg (V(max)) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 A resolution. The 3D structure of an endo-1,4-beta-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.
Biochemistry (Mosc). 2012 Oct;77(10):1190-8. doi: 10.1134/S0006297912100112.   PUBMED

Use of noncrystallographic symmetry for automated model building at medium to low resolution.
Wiegels T, Lamzin VS
A novel method is presented for the automatic detection of noncrystallographic symmetry (NCS) in macromolecular crystal structure determination which does not require the derivation of molecular masks or the segmentation of density. It was found that throughout structure determination the NCS-related parts may be differently pronounced in the electron density. This often results in the modelling of molecular fragments of variable length and accuracy, especially during automated model-building procedures. These fragments were used to identify NCS relations in order to aid automated model building and refinement. In a number of test cases higher completeness and greater accuracy of the obtained structures were achieved, specifically at a crystallographic resolution of 2.3 A or poorer. In the best case, the method allowed the building of up to 15% more residues automatically and a tripling of the average length of the built fragments.
Acta Crystallogr D Biol Crystallogr. 2012 Apr;68(Pt 4):446-53. doi: 10.1107/S0907444911050712. Epub 2012 Mar 16.   PUBMED

Aspartate aminotransferase: bridging carbohydrate and energy metabolism in Plasmodium falciparum.
Wrenger C, Muller IB, Silber AM, Jordanova R, Lamzin VS, Groves MR
In this mini-review we briefly examine and summarize evidence on the role of the plasmodial aspartate aminotransferase (AspAT) of the malarial parasite. Recent data have provided information on the products of the purine salvage pathway as well as the glycolytic and oxidative phosphorylation pathways, suggesting that the reaction catalyzed by AspAT is an essential step in all these biochemical processes. While the biological role of the oxidative phosphorylation cycle still remains to be demonstrated, the presence of a single protein that is functional in multiple pathways (i.e. amino acid/purine/pyrimidine biosynthesis and carbohydrate metabolism) provides a high potential for the development of novel strategies to combat the spread of multi-drug resistant malaria.
Curr Drug Metab. 2012 Mar;13(3):332-6.   PUBMED

Fragmentation-tree density representation for crystallographic modelling of bound ligands.
Langer GG, Evrard GX, Carolan CG, Lamzin VS
The identification and modelling of ligands into macromolecular models is important for understanding molecule's function and for designing inhibitors to modulate its activities. We describe new algorithms for the automated building of ligands into electron density maps in crystal structure determination. Location of the ligand-binding site is achieved by matching numerical shape features describing the ligand to those of density clusters using a "fragmentation-tree" density representation. The ligand molecule is built using two distinct algorithms exploiting free atoms with inter-atomic connectivity and Metropolis-based optimisation of the conformational state of the ligand, producing an ensemble of structures from which the final model is derived. The method was validated on several thousand entries from the Protein Data Bank. In the majority of cases, the ligand-binding site could be correctly located and the ligand model built with a coordinate accuracy of better than 1 A. We anticipate that the method will be of routine use to anyone modelling ligands, lead compounds or even compound fragments as part of protein functional analyses or drug design efforts.
J Mol Biol. 2012 Jun 8;419(3-4):211-22. doi: 10.1016/j.jmb.2012.03.012. Epub 2012 Mar 23.   PUBMED


Crystal structure of D-serine dehydratase from Escherichia coli.
Urusova DV, Isupov MN, Antonyuk S, Kachalova GS, Obmolova G, Vagin AA, Lebedev AA, Burenkov GP, Dauter Z, Bartunik HD, Lamzin VS, Melik-Adamyan WR, Mueller TD, Schnackerz KD
D-Serine dehydratase from Escherichia coli is a member of the beta-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97A-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55A resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the beta-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.
Biochim Biophys Acta. 2012 Mar;1824(3):422-32. doi: 10.1016/j.bbapap.2011.10.017. Epub 2011 Nov 27.   PUBMED

Amphiphilic nanotubes in the crystal structure of a biosurfactant protein hydrophobin HFBII.
Kallio JM, Rouvinen J
Atomic scale experimental data by X-ray crystallography have been collected on an amphiphilic protein nanotube, consisting of a biosurfactant protein Trichoderma reesei hydrophobin HFBII.
Chem Commun (Camb). 2011 Sep 21;47(35):9843-5. doi: 10.1039/c1cc13139g. Epub 2011 Aug 2.   PUBMED

On the routine use of soft X-rays in macromolecular crystallography. Part V. Molecular replacement and anomalous scattering.
Unge J, Mueller-Dieckmann C, Panjikar S, Tucker PA, Lamzin VS, Weiss MS
Currently, about two thirds of all new macromolecular structures are determined by molecular replacement. In general the method works reliably, but it reaches its limits when the search model differs too much from the target structure in terms of coordinate deviations or completeness. Since anomalously scattering substructures are better conserved than the overall structure, these substructures and the corresponding anomalous intensity differences can be utilized to enhance the performance of molecular-replacement approaches. It is demonstrated that the combined and concomitant use of structure-factor amplitudes and anomalous differences constitutes a promising approach to push the limits of molecular replacement and to make more structures amenable to structure solution by this technique.
Acta Crystallogr D Biol Crystallogr. 2011 Aug;67(Pt 8):729-38. doi: 10.1107/S0907444911024887. Epub 2011 Jul 12.   PUBMED

Crystallization and preliminary X-ray crystallographic studies of an oligomeric species of a refolded C39 peptidase-like domain of the Escherichia coli ABC transporter haemolysin B.
Schwarz CK, Tschapek B, Jumpertz T, Jenewein S, Lecher J, Willbold D, Panjikar S, Holland IB, Smits SH, Schmitt L
The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 May 1;67(Pt 5):630-3. doi: 10.1107/S1744309111010876. Epub 2011 Apr 28.   PUBMED

Additional phase information from UV damage of selenomethionine labelled proteins.
de Sanctis D, Tucker PA, Panjikar S
Currently, selenium is the most widely used phasing vehicle for experimental phasing, either by single anomalous scattering or multiple-wavelength anomalous dispersion (MAD) procedures. The use of the single isomorphous replacement anomalous scattering (SIRAS) phasing procedure with selenomethionine containing proteins is not so commonly used, as it requires isomorphous native data. Here it is demonstrated that isomorphous differences can be measured from intensity changes measured from a selenium labelled protein crystal before and after UV exposure. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment. The phasing procedure for two selenomethionine proteins, the feruloyl esterase module of xylanase 10B from Clostridium thermocellum and the Mycobacterium tuberculosis chorismate synthase, have been investigated using datasets collected near the absorption edge of selenium before and after UV radiation. The utility of UV radiation in measuring radiation damage data for isomorphous differences is highlighted and it is shown that, after such measurements, the UV-RIPAS procedure yields comparable phase sets with those obtained from the conventional MAD procedure. The results presented are encouraging for the development of alternative phasing approaches for selenomethionine proteins in difficult cases.
J Synchrotron Radiat. 2011 May;18(Pt 3):374-80. doi: 10.1107/S0909049511004092. Epub 2011 Mar 11.   PUBMED

Crystal structure of small protein crambin at 0.48 A resolution.
Schmidt A, Teeter M, Weckert E, Lamzin VS
With the development of highly brilliant and extremely intense synchrotron X-ray sources, extreme high-resolution limits for biological samples are now becoming attainable. Here, a study is presented that sets the record in crystallographic resolution for a biological macromolecule. The structure of the small protein crambin was determined to 0.48 A resolution on the PETRA II ring before its conversion to a dedicated synchrotron-radiation source. The results reveal a wealth of details in electron density and demonstrate the possibilities that are potentially offered by a high-energy source. The question now arises as to what the true limits are in terms of what can be seen at such high resolution. From what can be extrapolated from the results using crystals of crambin, this limit would be at approximately 0.40 A, which approaches that for smaller compounds.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Apr 1;67(Pt 4):424-8. doi: 10.1107/S1744309110052607. Epub 2011 Mar 24.   PUBMED

Structural characterization of the multidomain regulatory protein Rv1364c from Mycobacterium tuberculosis.
King-Scott J, Konarev PV, Panjikar S, Jordanova R, Svergun DI, Tucker PA
The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent sigma factor, sigma(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response sigma factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-sigma(F) like) domain and a C-terminal anti-sigma(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6 A) and a phosphatase/kinase dual domain construct (at 2.6 A) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding, affects the accessibility of the serine of the C-terminal, anti-sigma(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of sigma(F).
Structure. 2011 Jan 12;19(1):56-69. doi: 10.1016/j.str.2010.11.010.   PUBMED

Single isomorphous replacement phasing of selenomethionine-containing proteins using UV-induced radiation damage.
Panjikar S, Mayerhofer H, Tucker PA, Mueller-Dieckmann J, de Sanctis D
The most commonly used heavy-atom derivative, selenium, requires the use of a tunable beamline to access the Se K edge for experimental phasing using anomalous diffraction methods, whereas X-ray diffraction experiments for selenium-specific ultraviolet radiation-damage-induced phasing can be performed on fixed-wavelength beamlines or even using in-house X-ray sources. Several nonredundant X-ray diffraction data sets were collected from three different selenomethionine (Mse) derivatized protein crystals at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation using 266 nm lasers of flux density 1.7 x 10(1)(5) photons s(-)(1) mm(-)(2) for 10-50 min. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation-damage-induced phasing (RIP) technique. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing when no tunable X-ray source is available. The behaviour of Mse derivatives of various proteins provides novel insights and an initial basis for understanding the mechanism of selenium-specific UV radiation damage.
Acta Crystallogr D Biol Crystallogr. 2011 Jan;67(Pt 1):32-44. doi: 10.1107/S090744491004299X. Epub 2010 Dec 16.   PUBMED


Structural basis for the oxidation of protein-bound sulfur by the sulfur cycle molybdohemo-enzyme sulfane dehydrogenase SoxCD.
Zander U, Faust A, Klink BU, de Sanctis D, Panjikar S, Quentmeier A, Bardischewsky F, Friedrich CG, Scheidig AJ
The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an alpha(2)beta(2) heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D(1) and D(2). SoxCD(1) misses the heme-2 domain D(2) and is catalytically as active as SoxCD. The crystal structure of SoxCD(1) was solved at 1.33 A. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD(1) substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD(1). For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD(1) the structures of all key enzymes of the Sox cycle are known with atomic resolution.
J Biol Chem. 2011 Mar 11;286(10):8349-60. doi: 10.1074/jbc.M110.193631. Epub 2010 Dec 8.   PUBMED

Crystal structures of Trichoderma reesei beta-galactosidase reveal conformational changes in the active site.
Maksimainen M, Hakulinen N, Kallio JM, Timoharju T, Turunen O, Rouvinen J
We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) beta-galactosidase (Tr-beta-gal) at a 1.2A resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4A resolutions, respectively. Tr-beta-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-beta-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-beta-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.
J Struct Biol. 2011 Apr;174(1):156-63. doi: 10.1016/j.jsb.2010.11.024. Epub 2010 Dec 3.   PUBMED

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum.
Wrenger C, Muller IB, Schifferdecker AJ, Jain R, Jordanova R, Groves MR
Aspartate aminotransferases (AspATs; EC catalyze the conversion of aspartate and alpha-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 A. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria.
J Mol Biol. 2011 Jan 28;405(4):956-71. doi: 10.1016/j.jmb.2010.11.018. Epub 2010 Nov 16.   PUBMED

Quantitive evaluation of macromolecular crystallization experiments using 1,8-ANS fluorescence.
Watts D, Muller-Dieckmann J, Tsakanova G, Lamzin VS, Groves MR
Modern X-ray structure analysis and advances in high-throughput robotics have allowed a significant increase in the number of conditions screened for a given sample volume. An efficient evaluation of the increased amount of crystallization trials in order to identify successful experiments is now urgently required. A novel approach is presented for the visualization of crystallization experiments using fluorescence from trace amounts of a nonspecific dye. The fluorescence images obtained strongly contrast protein crystals against other phenomena, such as precipitation and phase separation. Novel software has been developed to quantitatively evaluate the crystallization outcome based on a biophysical metric correlated with voxel protein concentration. In >1500 trials, 85.6% of the successful crystallization experiments were correctly identified, yielding a 50% reduction in the number of 'missed hits' compared with current automated approaches. The use of the method in the crystallization of three previously uncharacterized proteins from the malarial parasite Plasmodium falciparum is further demonstrated.
Acta Crystallogr D Biol Crystallogr. 2010 Aug;66(Pt 8):901-8. doi: 10.1107/S0907444910020664. Epub 2010 Jul 10.   PUBMED

A moment invariant for evaluating the chirality of three-dimensional objects.
Hattne J, Lamzin VS
Chirality is an important feature of three-dimensional objects and a key concept in chemistry, biology and many other disciplines. However, it has been difficult to quantify, largely owing to computational complications. Here we present a general chirality measure, called the chiral invariant (CI), which is applicable to any three-dimensional object containing a large amount of data. The CI distinguishes the hand of the object and quantifies the degree of its handedness. It is invariant to the translation, rotation and scale of the object, and tolerant to a modest amount of noise in the experimental data. The invariant is expressed in terms of moments and can be computed in almost no time. Because of its universality and computational efficiency, the CI is suitable for a wide range of pattern-recognition problems. We demonstrate its applicability to molecular atomic models and their electron density maps. We show that the occurrence of the conformations of the macromolecular polypeptide backbone is related to the value of the CI of the constituting peptide fragments. We also illustrate how the CI can be used to assess the quality of a crystallographic electron density map.
J R Soc Interface. 2011 Jan 6;8(54):144-51. doi: 10.1098/rsif.2010.0297. Epub 2010 Aug 4.   PUBMED

Purification, crystallization and preliminary X-ray analysis of the aspartate aminotransferase of Plasmodium falciparum.
Jain R, Jordanova R, Muller IB, Wrenger C, Groves MR
Aspartate aminotransferases (EC catalyse the conversion of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate in a reversible manner. Thus, the aspartate aminotransferase of Plasmodium falciparum (PfAspAT) plays a central role in the transamination of amino acids. Recent findings suggest that PfAspAT may also play a pivotal role in energy metabolism and the de novo biosynthesis of pyrimidines. While therapeutics based upon the inhibition of other proteins in these pathways are already used in the treatment of malaria, the advent of multidrug-resistant strains has limited their efficacy. The presence of PfAspAT in these pathways may offer additional opportunities for the development of novel therapeutics. In order to gain a deeper understanding of the function and role of PfAspAT, it has been expressed and purified to homogeneity. The successful crystallization of PfAspAT, the collection of a 2.8 A diffraction data set and initial attempts to solve the structure using molecular replacement are reported.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Apr 1;66(Pt 4):409-12. doi: 10.1107/S1744309110003933. Epub 2010 Mar 31.   PUBMED

Internal motion in protein crystal structures.
Schmidt A, Lamzin VS
The binding states of the substrates and the environment have significant influence on protein motion. We present the analysis of such motion derived from anisotropic atomic displacement parameters (ADPs) in a set of atomic resolution protein structures. Local structural motion caused by ligand binding as well as functional loops showing cooperative patterns of motion could be inferred. The results are in line with proposed protonation states, hydrogen bonding patterns and the location of distinctly flexible regions: we could locate the mobile active site loop in a virus integrase, distinguish the subdomains in RNAse A and hydroxynitrile lyase, and reconstruct the molecular architecture in a xylanase. We demonstrate that the ADP-based motion analysis provides information at high level of detail and that the structural changes needed for substrate attachment or release may be derived from single X-ray structures.
Protein Sci. 2010 May;19(5):944-53. doi: 10.1002/pro.371.   PUBMED


The vitamin B1 metabolism of Staphylococcus aureus is controlled at enzymatic and transcriptional levels.
Muller IB, Bergmann B, Groves MR, Couto I, Amaral L, Begley TP, Walter RD, Wrenger C
Vitamin B1 is in its active form thiamine pyrophosphate (TPP), an essential cofactor for several key enzymes in the carbohydrate metabolism. Mammals must salvage this crucial nutrient from their diet in order to complement the deficiency of de novo synthesis. In the human pathogenic bacterium Staphylococcus aureus, two operons were identified which are involved in vitamin B1 metabolism. The first operon encodes for the thiaminase type II (TenA), 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (ThiD), 5-(2-hydroxyethyl)-4-methylthiazole kinase (ThiM) and thiamine phosphate synthase (ThiE). The second operon encodes a phosphatase, an epimerase and the thiamine pyrophosphokinase (TPK). The open reading frames of the individual operons were cloned, their corresponding proteins were recombinantly expressed and biochemically analysed. The kinetic properties of the enzymes as well as the binding of TPP to the in vitro transcribed RNA of the proposed operons suggest that the vitamin B1 homeostasis in S. aureus is strongly regulated at transcriptional as well as enzymatic levels.
PLoS One. 2009 Nov 3;4(11):e7656. doi: 10.1371/journal.pone.0007656.   PUBMED

Fatty acid- and retinoid-binding proteins have distinct binding pockets for the two types of cargo.
Jordanova R, Groves MR, Kostova E, Woltersdorf C, Liebau E, Tucker PA
Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The crystal structure of C. elegans FAR-7 is the first structure of a FAR protein, and it exhibits a novel fold. It differs radically from the mammalian fatty acid-binding proteins and has two ligand binding pockets joined by a surface groove. The first can accommodate the aliphatic chain of fatty acids, whereas the second can accommodate the bulkier retinoids. In addition to demonstrating lipid binding by fluorescence spectroscopy, we present evidence that retinol binding is positively regulated by casein kinase II phosphorylation at a conserved site near the bottom of the second pocket. far-7::GFP (green fluorescent protein) expression shows that it is localized in the head hypodermal syncytia and the excretory cell but that this localization changes under starvation conditions. In conclusion, our study provides the basic structural and functional information for investigation of inhibitors of lipid binding by FAR proteins.
J Biol Chem. 2009 Dec 18;284(51):35818-26. doi: 10.1074/jbc.M109.022731.   PUBMED

On the combination of molecular replacement and single-wavelength anomalous diffraction phasing for automated structure determination.
Panjikar S, Parthasarathy V, Lamzin VS, Weiss MS, Tucker PA
A combination of molecular replacement and single-wavelength anomalous diffraction phasing has been incorporated into the automated structure-determination platform Auto-Rickshaw. The complete MRSAD procedure includes molecular replacement, model refinement, experimental phasing, phase improvement and automated model building. The improvement over the standard SAD or MR approaches is illustrated by ten test cases taken from the JCSG diffraction data-set database. Poor MR or SAD phases with phase errors larger than 70 degrees can be improved using the described procedure and a large fraction of the model can be determined in a purely automatic manner from X-ray data extending to better than 2.6 A resolution.
Acta Crystallogr D Biol Crystallogr. 2009 Oct;65(Pt 10):1089-97. doi: 10.1107/S0907444909029643. Epub 2009 Sep 16.   PUBMED

Interpretation of very low resolution X-ray electron-density maps using core objects.
Heuser P, Langer GG, Lamzin VS
A novel approach to obtaining structural information from macromolecular X-ray data extending to resolutions as low as 20 A is presented. Following a simple map-segmentation procedure, the approximate shapes of the domains forming the structure are identified. A pattern-recognition comparative analysis of these shapes and those derived from the structures of domains from the PDB results in candidate structural models that can be used for a fit into the density map. It is shown that the placed candidate models can be employed for subsequent phase extension to higher resolution.
Acta Crystallogr D Biol Crystallogr. 2009 Jul;65(Pt 7):690-6. doi: 10.1107/S090744490901991X. Epub 2009 Jun 20.   PUBMED

Structure of native laccase from Trametes hirsuta at 1.8 A resolution.
Polyakov KM, Fedorova TV, Stepanova EV, Cherkashin EA, Kurzeev SA, Strokopytov BV, Lamzin VS, Koroleva OV
This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.
Acta Crystallogr D Biol Crystallogr. 2009 Jun;65(Pt 6):611-7. doi: 10.1107/S0907444909011950. Epub 2009 May 15.   PUBMED

High-resolution structural analysis of a novel octaheme cytochrome c nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens.
Polyakov KM, Boyko KM, Tikhonova TV, Slutsky A, Antipov AN, Zvyagilskaya RA, Popov AN, Bourenkov GP, Lamzin VS, Popov VO
Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.
J Mol Biol. 2009 Jun 26;389(5):846-62. doi: 10.1016/j.jmb.2009.04.037. Epub 2009 Apr 23.   PUBMED

Mobility of the conserved glycine 155 is required for formation of the active plasmodial Pdx1 dodecamer.
Knockel J, Jordanova R, Muller IB, Wrenger C, Groves MR
BACKGROUND: Vitamin B6 synthesis requires a functional Pdx1 assembly that is dodecameric in vivo. We have previously shown that mutation of a catalytic lysine in the plasmodial Pdx1 protein results in a protein that is both inactive and hexameric in vitro. METHODS: Static and dynamic light scattering, circular dichroism, co-purification and enzyme assays are used to investigate the role of a glycine conserved in all Pdx1 family members. RESULTS: Static light scattering indicates that a glycine to alanine mutant is present as a hexamer in vitro. Subsequent circular dichroism experiments demonstrate that a significant change in secondary structure content is induced by this mutation. However, this mutant is still competent to bind and support Pdx2 activity. CONCLUSIONS: As the mutated glycine occupies an unrestricted region of the Ramachandran plot the additional stereo-chemical restrictions imposed on alanine residues strongly support our hypothesis that significant structural rearrangement of Pdx1 is required during the transition from hexamer to dodecamer. GENERAL SIGNIFICANCE: The presented results demonstrate that reduction in the mobility of this region in Pdx1 proteins is required for formation of the in vivo dodecamer, negatively affecting the activity of Pdx1, opening the possibility of allosteric Pdx1 inhibitors.
Biochim Biophys Acta. 2009 May;1790(5):347-50. doi: 10.1016/j.bbagen.2009.02.016. Epub 2009 Mar 9.   PUBMED

The three-component signalling system HbpS-SenS-SenR as an example of a redox sensing pathway in bacteria.
Ortiz de Orue Lucana D, Groves MR
The two-component system SenS-SenR and the extracellular HbpS protein of the cellulose degrader Streptomyces reticuli have been shown to act in concert as a novel system which detects redox stress. In vivo and in vitro experiments have led to the hypothesis that HbpS binds and degrades heme, communicating the extracellular presence of heme and oxidative stress to the membrane-embedded sensor histidine kinase SenS via a bound iron. The response regulator SenR would then up-regulate downstream signalling cascades, leading to the appropriate gene expression levels for bacterial survival in an oxidative environment. Sequence analysis has shown that homologs of HbpS and SenS-SenR exist in a number of ecologically and medically relevant bacterial species, suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to both Gram-negative and Gram-positive bacteria. The presented report reviews the current knowledge of the function of this novel protein family consisting of an accessory protein and its cognate two-component system, which could be more properly described as a three-component system.
Amino Acids. 2009 Sep;37(3):479-86. doi: 10.1007/s00726-009-0260-9. Epub 2009 Mar 4.   PUBMED

The oligomeric assembly of the novel haem-degrading protein HbpS is essential for interaction with its cognate two-component sensor kinase.
Ortiz de Orue Lucana D, Bogel G, Zou P, Groves MR
HbpS, a novel protein of previously unknown function from Streptomyces reticuli, is up-regulated in response to haemin- and peroxide-based oxidative stress and interacts with the SenS/SenR two-component signal transduction system. In this study, we report the high-resolution crystal structures (2.2 and 1.6 A) of octomeric HbpS crystallized in the presence and in the absence of haem and demonstrate that iron binds to surface-exposed lysine residues of an octomeric assembly. Based on an analysis of the crystal structures, we propose that the iron atom originates from the haem group and report subsequent biochemical experiments that demonstrate that HbpS possesses haem-degrading activity in vitro. Further examination of the crystal structures has identified amino acids that are essential for assembly of the octomer. The role of these residues is confirmed by biophysical experiments. Additionally, we show that while the octomeric assembly state of HbpS is not essential for haem-degrading activity, the assembly of HbpS is required for its interaction with the cognate sensor kinase, SenS. Homologs of HbpS and SenS/SenR have been identified in a number of medically and ecologically relevant bacterial species (including Vibrio cholerae, Klebsiella pneumoniae, Corynebacterium diphtheriae, Arthrobacter aurescens and Pseudomonas putida), suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to Gram-negative and Gram-positive bacteria. Thus, the data presented provide the first insight into the function of a novel protein family and an example of an iron-mediated interaction between an accessory protein and its cognate two-component sensor kinase.
J Mol Biol. 2009 Mar 6;386(4):1108-22.   PUBMED


Crystallization and preliminary X-ray analysis of the Thermoplasma acidophilum 20S proteasome in complex with protein substrates.
Felderer K, Groves M, Diez J, Pohl E, Witt S
The 20S proteasome is a 700 kDa barrel-shaped proteolytic complex that is traversed by an internal channel which widens into three cavities: two antechambers and one central chamber. Entrance to the complex is restricted by the narrow opening of the channel, which only allows unfolded substrates to reach the active sites located within the central cavity. The X-ray structures of 20S proteasomes from different organisms with and without inhibitors bound have led to a detailed knowledge of their structure and proteolytic function. Nevertheless, the mechanisms that underlie substrate translocation into the 20S proteasome and the role of the antechambers remain elusive. To investigate putative changes within the proteasome that occur during substrate translocation, ;host-guest' complexes between the Thermoplasma acidophilum 20S proteasomes and either cytochrome c (cyt c) or green fluorescent protein (GFP) were produced and crystallized. Orthorhombic crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 116, b = 207, c = 310 A (cyt c) and a = 116, b = 206, c = 310 A (GFP), were formed and X-ray diffraction data were collected to 3.4 A (cyt c) and 3.8 A (GFP) resolution.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Oct 1;64(Pt 10):899-902. doi: 10.1107/S1744309108026791. Epub 2008 Sep 30.   PUBMED

Pattern-recognition-based detection of planar objects in three-dimensional electron-density maps.
Hattne J, Lamzin VS
A pattern-recognition-based method for the detection of planar objects in protein or DNA/RNA crystal structure determination is described. The procedure derives a set of rotation-invariant numeric features from local regions in the asymmetric unit of a crystallographic electron-density map. These features, primarily moments of various orders, capture different aspects of the local shape of objects in the electron density. Feature classification is achieved using a linear discriminant that is trained to optimize the contrast between planar and nonplanar objects. In five selected test cases with X-ray data spanning 2.0-3.0 A resolution, the procedure identified the correct location and orientation for almost all of the double-ring and a majority of the single-ring planar groups. The accuracy of the location of the plane centres is of the order of 0.5 A, even in moderately noisy density maps.
Acta Crystallogr D Biol Crystallogr. 2008 Aug;D64(Pt 8):834-42. doi: 10.1107/S0907444908014327. Epub 2008 Jul 17.   PUBMED

Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.
Langer G, Cohen SX, Lamzin VS, Perrakis A
ARP/wARP is a software suite to build macromolecular models in X-ray crystallography electron density maps. Structural genomics initiatives and the study of complex macromolecular assemblies and membrane proteins all rely on advanced methods for 3D structure determination. ARP/wARP meets these needs by providing the tools to obtain a macromolecular model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water molecules. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.
Nat Protoc. 2008;3(7):1171-9. doi: 10.1038/nprot.2008.91.   PUBMED

Methods for protein characterization by mass spectrometry, thermal shift (ThermoFluor) assay, and multiangle or static light scattering.
Nettleship JE, Brown J, Groves MR, Geerlof A
Mass spectrometry (MS) is widely used within structural and functional proteomics for a variety of tasks including protein quality assessment, identification, and characterization. MS is used routinely for the determination of the total mass of proteins, including N-glycosylated proteins, analysis of selenomethionine incorporation, crystal content verification, and analysis of N-glycosylation site occupancy. Protocols for sample preparation, data collection, and analysis are given.A recent development is the fluorescence-based thermal shift (ThermoFluor) assay. It uses an environmentally sensitive dye, Sypro Orange, to monitor the thermal stability of a protein and investigate factors (e.g., buffers, additives, and ligands) affecting this stability. This chapter describes the application of this method using a 96-condition in-house screen. The measurements are performed on a commercially available real-time PCR machine. Multiangle or static light scattering (SLS) is a very powerful technique to determine the conformational state of proteins in solution, especially when used in combination with size exclusion chromatography (SEC). In the authors' experimental set-up the SLS detector is connected in-line to a standard protein purification machine (e.g., the Akta Purifier) equipped with an analytical SEC column. The data collection and analysis are performed using commercial software.
Methods Mol Biol. 2008;426:299-318. doi: 10.1007/978-1-60327-058-8_19.   PUBMED

Atomic resolution crystal structures and quantum chemistry meet to reveal subtleties of hydroxynitrile lyase catalysis.
Schmidt A, Gruber K, Kratky C, Lamzin VS
Hydroxynitrile lyases are versatile enzymes that enantiospecifically cope with cyanohydrins, important intermediates in the production of various agrochemicals or pharmaceuticals. We determined four atomic resolution crystal structures of hydroxynitrile lyase from Hevea brasiliensis: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate. We observed distinct distance changes among the active site residues related to proton shifts upon substrate binding. The combined use of crystallography and ab initio quantum chemical calculations allowed the determination of the protonation states in the enzyme active site. We show that His(235) of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations. We also found evidence for the considerable pK(a) shifts that had been hypothesized earlier. We envision that this knowledge can be used to enhance the catalytic properties and the stability of the enzyme for industrial production of enantiomerically pure cyanohydrins.
J Biol Chem. 2008 Aug 1;283(31):21827-36. doi: 10.1074/jbc.M801056200. Epub 2008 Jun 4.   PUBMED

Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli.
Zou P, Groves MR, Viale-Bouroncle SD, Ortiz de Orue Lucana D
Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe3+ oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS-SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS-SenR, which regulates the expression of the catalase-peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2(1)3, with a cell edge of 152.5 A. Diffraction data were recorded to a maximal resolution of 2.25 A and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 May 1;64(Pt 5):386-90. doi: 10.1107/S1744309108008348. Epub 2008 Apr 5.   PUBMED

A knowledge-driven approach for crystallographic protein model completion.
Joosten K, Cohen SX, Emsley P, Mooij W, Lamzin VS, Perrakis A
One of the most cumbersome and time-demanding tasks in completing a protein model is building short missing regions or ;loops'. A method is presented that uses structural and electron-density information to build the most likely conformations of such loops. Using the distribution of angles and dihedral angles in pentapeptides as the driving parameters, a set of possible conformations for the C(alpha) backbone of loops was generated. The most likely candidate is then selected in a hierarchical manner: new and stronger restraints are added while the loop is built. The weight of the electron-density correlation relative to geometrical considerations is gradually increased until the most likely loop is selected on map correlation alone. To conclude, the loop is refined against the electron density in real space. This is started by using structural information to trace a set of models for the C(alpha) backbone of the loop. Only in later steps of the algorithm is the electron-density correlation used as a criterion to select the loop(s). Thus, this method is more robust in low-density regions than an approach using density as a primary criterion. The algorithm is implemented in a loop-building program, Loopy, which can be used either alone or as part of an automatic building cycle. Loopy can build loops of up to 14 residues in length within a couple of minutes. The average root-mean-square deviation of the C(alpha) atoms in the loops built during validation was less than 0.4 A. When implemented in the context of automated model building in ARP/wARP, Loopy can increase the completeness of the built models.
Acta Crystallogr D Biol Crystallogr. 2008 Apr;64(Pt 4):416-24. doi: 10.1107/S0907444908001558. Epub 2008 Mar 19.   PUBMED

Crystal structure of YagE, a putative DHDPS-like protein from Escherichia coli K12.
Manicka S, Peleg Y, Unger T, Albeck S, Dym O, Greenblatt HM, Bourenkov G, Lamzin V, Krishnaswamy S, Sussman JL
no abstract available
Proteins. 2008 Jun;71(4):2102-8. doi: 10.1002/prot.22023.   PUBMED

The assembly of the plasmodial PLP synthase complex follows a defined course.
Muller IB, Knockel J, Groves MR, Jordanova R, Ealick SE, Walter RD, Wrenger C
BACKGROUND: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.
PLoS One. 2008 Mar 19;3(3):e1815. doi: 10.1371/journal.pone.0001815.   PUBMED


ARP/wARP and molecular replacement: the next generation.
Cohen SX, Ben Jelloul M, Long F, Vagin A, Knipscheer P, Lebbink J, Sixma TK, Lamzin VS, Murshudov GN, Perrakis A
Automatic iterative model (re-)building, as implemented in ARP/wARP and its new control system flex-wARP, is particularly well suited to follow structure solution by molecular replacement. More than 100 molecular-replacement solutions automatically solved by the BALBES software were submitted to three standard protocols in flex-wARP and the results were compared with final models from the PDB. Standard metrics were gathered in a systematic way and enabled the drawing of statistical conclusions on the advantages of each protocol. Based on this analysis, an empirical estimator was proposed that predicts how good the final model produced by flex-wARP is likely to be based on the experimental data and the quality of the molecular-replacement solution. To introduce the differences between the three flex-wARP protocols (keeping the complete search model, converting it to atomic coordinates but ignoring atom identities or using the electron-density map calculated from the molecular-replacement solution), two examples are also discussed in detail, focusing on the evolution of the models during iterative rebuilding. This highlights the diversity of paths that the flex-wARP control system can employ to reach a nearly complete and accurate model while actually starting from the same initial information.
Acta Crystallogr D Biol Crystallogr. 2008 Jan;64(Pt 1):49-60. doi: 10.1107/S0907444907047580. Epub 2007 Dec 5.   PUBMED

High-resolution structures of formate dehydrogenase from Candida boidinii.
Schirwitz K, Schmidt A, Lamzin VS
The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD(+)-dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific activity, has been targeted by enzyme engineering for several years. Optimization by mutagenesis studies was performed based on physiological studies and structure modeling. However, X-ray structural information has been required in order to clarify the enzymatic mechanism and to enhance the effectiveness and operational stability of enzymatic cofactor regenerators in biocatalytic enantiomer synthesis as well as to explain the observed biochemical differences between yeast and bacterial FDH. We designed two single-point mutants in CbFDH using an adapted surface engineering approach, and this allowed crystals suitable for high-resolution X-ray structural studies to be obtained. The mutations improved the crystallizability of the protein and also the catalytic properties and the stability of the enzyme. With these crystal structures, we explain the observed differences from both sources, and form the basis for further rational mutagenesis studies.
Protein Sci. 2007 Jun;16(6):1146-56. doi: 10.1110/ps.062741707.   PUBMED

From atoms to proteins.
Schmidt A, Lamzin VS
The investigation of biological macromolecules and the characteristics that determine their function has been of particular interest over the last decades. Here we overview some modern approaches for making the most of the 3-D protein structural information, with a distinctive emphasis on macromolecular crystallography and complementary techniques used to establish the structure-function relationship. A tight link between the biology of the cellular processes and the underlying chemistry of protein function governs the flow of the presented material. The reader will be lead through the basic principles of protein structure analysis and the means to capture the characteristics that portray the function. The techniques exploiting high-resolution data and allowing quantification of molecular motion and structure-activity relationship are given particular attention.
Cell Mol Life Sci. 2007 Aug;64(15):1959-69. doi: 10.1007/s00018-007-7195-7.   PUBMED

Structural evidence for a ligand coordination switch in liver alcohol dehydrogenase.
Meijers R, Adolph HW, Dauter Z, Wilson KS, Lamzin VS, Cedergren-Zeppezauer ES
The use of substrate analogues as inhibitors provides a way to understand and manipulate enzyme function. Here we report two 1 A resolution crystal structures of liver alcohol dehydrogenase in complex with NADH and two inhibitors: dimethyl sulfoxide and isobutyramide. Both structures present a dynamic state of inhibition. In the dimethyl sulfoxide complex structure, the inhibitor is caught in transition on its way to the active site using a flash-freezing protocol and a cadmium-substituted enzyme. One inhibitor molecule is partly located in the first and partly in the second coordination sphere of the active site metal. A hydroxide ion bound to the active site metal lies close to the pyridine ring of NADH, which is puckered in a twisted boat conformation. The cadmium ion is coordinated by both the hydroxide ion and the inhibitor molecule, providing structural evidence of a coordination switch at the active site metal ion. The structure of the isobutyramide complex reveals the partial formation of an adduct between the isobutyramide inhibitor and NADH. It provides evidence of the contribution of a shift from the keto to the enol tautomer during aldehyde reduction. The different positions of the inhibitors further refine the knowledge of the dynamics of the enzyme mechanism and explain how the crowded active site can facilitate the presence of a substrate and a metal-bound hydroxide ion.
Biochemistry. 2007 May 8;46(18):5446-54. doi: 10.1021/bi6023594. Epub 2007 Apr 13.   PUBMED

A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye.
Groves MR, Muller IB, Kreplin X, Muller-Dieckmann J
A technique is described whereby the addition of low concentrations (millimolar to micromolar) of the fluorescent dye 1,8-ANS to the protein solution prior to crystallization results in crystallization experiments in which protein crystals are strongly contrasted above background artifacts when exposed to low-intensity UV radiation. As 1,8-ANS does not covalently modify the protein sample, no further handling or purification steps are necessary. The system has been tested on a wide variety of protein samples and it has been shown that the addition of 1,8-ANS has no discernible effect on the crystallization frequencies or crystallization conditions of these proteins. As 1,8-ANS interacts with a wide variety of proteins, this is proposed to be a general solution for the automated classification of protein crystallization images and the detection of protein crystals. The results also demonstrate the expected discrimination between salt and protein crystals, as well as allowing the straightforward identification of small crystals that grow in precipitate or under a protein skin.
Acta Crystallogr D Biol Crystallogr. 2007 Apr;63(Pt 4):526-35. doi: 10.1107/S0907444906056137. Epub 2007 Mar 16.   PUBMED


Assessment of automatic ligand building in ARP/wARP.
Evrard GX, Langer GG, Perrakis A, Lamzin VS
The efficiency of the ligand-building module of ARP/wARP version 6.1 has been assessed through extensive tests on a large variety of protein-ligand complexes from the PDB, as available from the Uppsala Electron Density Server. Ligand building in ARP/wARP involves two main steps: automatic identification of the location of the ligand and the actual construction of its atomic model. The first step is most successful for large ligands. The second step, ligand construction, is more powerful with X-ray data at high resolution and ligands of small to medium size. Both steps are successful for ligands with low to moderate atomic displacement parameters. The results highlight the strengths and weaknesses of both the method of ligand building and the large-scale validation procedure and help to identify means of further improvement.
Acta Crystallogr D Biol Crystallogr. 2007 Jan;63(Pt 1):108-17. doi: 10.1107/S0907444906023389. Epub 2006 Dec 13.   PUBMED

Automated detection and centring of cryocooled protein crystals.
Pothineni SB, Strutz T, Lamzin VS
A novel method is presented for the automated recognition of cryocooled macromolecular crystals. The method uses several texture-based image-processing algorithms for automated crystal centring, which are able to cope with a variety of crystal morphologies and illumination conditions. The results combined from different algorithms, together with their estimated standard uncertainties, provide a robust determination of the crystal location and allow an internal assessment of their reliability. The method was coded within the software XREC and showed a good performance on 104 sets of images from various beamlines.
Acta Crystallogr D Biol Crystallogr. 2006 Nov;62(Pt 11):1358-68. doi: 10.1107/S0907444906031672. Epub 2006 Oct 18.   PUBMED

Purification, crystallization and preliminary X-ray study of the fungal laccase from Cerrena maxima.
Lyashenko AV, Zhukhlistova NE, Gabdoulkhakov AG, Zhukova YN, Voelter W, Zaitsev VN, Bento I, Stepanova EV, Kachalova GS, Koroleva OV, Cherkashyn EA, Tishkov VI, Lamzin VS, Schirwitz K, Morgunova EY, Betzel C, Lindley PF, Mikhailov AM
Laccases are members of the blue multi-copper oxidase family that oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and, following the transfer of four electrons, is reduced to two molecules of water. Crystals of the laccase from Cerrena maxima have been obtained and X-ray data were collected to 1.9 A resolution using synchrotron radiation. A preliminary analysis shows that the enzyme has the typical laccase structure and several carbohydrate sites have been identified. The carbohydrate chains appear to be involved in stabilization of the intermolecular contacts in the crystal structure, thus promoting the formation of well ordered crystals of the enzyme. Here, the results of an X-ray crystallographic study on the laccase from the fungus Cerrena maxima are reported. Crystals that diffract well to a resolution of at least 1.9 A (R factor = 18.953%; R(free) = 23.835; r.m.s.d. bond lengths, 0.06 A; r.m.s.d. bond angles, 1.07 degrees) have been obtained despite the presence of glycan moieties. The overall spatial organization of C. maxima laccase and the structure of its copper-containing active centre have been determined by the molecular-replacement method using the laccase from Trametes versicolor (Piontek et al., 2002) as a structural template. In addition, four glycan-binding sites were identified and the 1.9 A X-ray data were used to determine the previously unknown primary structure of this protein. The identity (calculated from sequence alignment) between the C. maxima laccase and the T. versicolor laccase is about 87%. Tyr196 and Tyr372 show significant extra density at the ortho positions and this has been interpreted in terms of NO(2) substituents.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Oct 1;62(Pt 10):954-7. doi: 10.1107/S1744309106036578. Epub 2006 Sep 19.   PUBMED

SPINE workshop on automated X-ray analysis: a progress report.
Bahar M, Ballard C, Cohen SX, Cowtan KD, Dodson EJ, Emsley P, Esnouf RM, Keegan R, Lamzin V, Langer G, Levdikov V, Long F, Meier C, Muller A, Murshudov GN, Perrakis A, Siebold C, Stein N, Turkenburg MG, Vagin AA, Winn M, Winter G, Wilson KS
The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.
Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1170-83. doi: 10.1107/S0907444906032197. Epub 2006 Sep 19.   PUBMED

X-ray structural studies of the fungal laccase from Cerrena maxima.
Lyashenko AV, Bento I, Zaitsev VN, Zhukhlistova NE, Zhukova YN, Gabdoulkhakov AG, Morgunova EY, Voelter W, Kachalova GS, Stepanova EV, Koroleva OV, Lamzin VS, Tishkov VI, Betzel C, Lindley PF, Mikhailov AM
Laccases are members of the blue multi-copper oxidase family. These enzymes oxidize substrate molecules by accepting electrons at a mononuclear copper centre and transferring them to a trinuclear centre. Dioxygen binds to the trinuclear centre and following the transfer of four electrons is reduced to two molecules of water. The X-ray structure of a laccase from Cerrena maxima has been elucidated at 1.9 A resolution using synchrotron data and the molecular replacement technique. The final refinement coefficients are Rcryst = 16.8% and Rfree = 23.0%, with root mean square deviations on bond lengths and bond angles of 0.015 A and 1.51 degrees , respectively. The type 1 copper centre has an isoleucine residue at the axial position and the "resting" state of the trinuclear centre comprises a single oxygen (OH) moiety asymmetrically disposed between the two type 3 copper ions and a water molecule attached to the type 2 ion. Several carbohydrate binding sites have been identified and the glycan chains appear to promote the formation of well-ordered crystals. Two tyrosine residues near the protein surface have been found in a nitrated state.
J Biol Inorg Chem. 2006 Nov;11(8):963-73. doi: 10.1007/s00775-006-0158-x. Epub 2006 Aug 30.   PUBMED

How to avoid premature decay of your macromolecular crystal: a quick soak for long life.
Kauffmann B, Weiss MS, Lamzin VS, Schmidt A
Radiation damage to biological samples is currently one of the major limiting factors in macromolecular X-ray crystallography, since it severely and irreversibly affects the quality of the data that can be obtained from a diffraction experiment. However, radiation damage can effectively be reduced by utilizing the electron and radical scavenging potential of certain small-molecule compounds. We propose an approach to protect macromolecular crystals prior to data collection by quick soaking with scavengers. This, in favorable cases, can more than double crystal lifetime in the X-ray beam. The approach has the potential to yield diffraction data of superior quality and hence to increase the amount of high-quality diffraction data and of structural information attainable from a single crystal.
Structure. 2006 Jul;14(7):1099-105. doi: 10.1016/j.str.2006.05.015.   PUBMED

Crystallization and preliminary X-ray analysis of cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens.
Boyko KM, Polyakov KM, Tikhonova TV, Slutsky A, Antipov AN, Zvyagilskaya RA, Bourenkov GP, Popov AN, Lamzin VS, Popov VO
A novel cytochrome c nitrite reductase (TvNiR) was isolated from the haloalkalophilic bacterium Thioalkalivibrio nitratireducens. The enzyme catalyses nitrite and hydroxylamine reduction, with ammonia as the only product of both reactions. It consists of 525 amino-acid residues and contains eight haems c. TvNiR crystals were grown by the hanging-drop vapour-diffusion technique. The crystals display cubic symmetry and belong to space group P2(1)3, with unit-cell parameter a = 194 A. A native data set was obtained to 1.5 A resolution. The structure was solved by the SAD technique using the data collected at the Fe absorption peak wavelength.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 Mar 1;62(Pt 3):215-7. doi: 10.1107/S174430910600296X. Epub 2006 Feb 10.   PUBMED


Extraction of functional motion in trypsin crystal structures.
Schmidt A, Lamzin VS
The analysis of anisotropic atomic displacement parameters for the direct extraction of functionally relevant motion from X-ray crystal structures of Fusarium oxysporum trypsin is presented. Several atomic resolution structures complexed with inhibitors or substrates and determined at different pH values and temperatures were investigated. The analysis revealed a breathing-like molecular motion conserved across trypsin structures from two organisms and three different crystal forms. Directional motion was observed suggesting a change of the width of the substrate-binding cleft and a change in the length of the specificity pocket. The differences in direction of motion across the structures are dependent on the mode of substrate or inhibitor binding and the chemical environment around the active-site residues. Together with the occurrence of multiple-residue conformers, they reflect spatial rearrangement throughout the deacylation pathway.
Acta Crystallogr D Biol Crystallogr. 2005 Aug;61(Pt 8):1132-9. doi: 10.1107/S0907444905016732. Epub 2005 Jul 20.   PUBMED

Auto-rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment.
Panjikar S, Parthasarathy V, Lamzin VS, Weiss MS, Tucker PA
The EMBL-Hamburg Automated Crystal Structure Determination Platform is a system that combines a number of existing macromolecular crystallographic computer programs and several decision-makers into a software pipeline for automated and efficient crystal structure determination. The pipeline can be invoked as soon as X-ray data from derivatized protein crystals have been collected and processed. It is controlled by a web-based graphical user interface for data and parameter input, and for monitoring the progress of structure determination. A large number of possible structure-solution paths are encoded in the system and the optimal path is selected by the decision-makers as the structure solution evolves. The processes have been optimized for speed so that the pipeline can be used effectively for validating the X-ray experiment at a synchrotron beamline.
Acta Crystallogr D Biol Crystallogr. 2005 Apr;61(Pt 4):449-57. doi: 10.1107/S0907444905001307. Epub 2005 Mar 24.   PUBMED


Towards complete validated models in the next generation of ARP/wARP.
Cohen SX, Morris RJ, Fernandez FJ, Ben Jelloul M, Kakaris M, Parthasarathy V, Lamzin VS, Kleywegt GJ, Perrakis A
The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated models comparable in quality to the result of intensive manual checking, rebuilding, hypothesis testing, refinement and validation cycles of an experienced crystallographer. In addition to the presentation of this control system, recent advances, ideas and future plans for improving the current model-building algorithms, especially for completing partially built models, are presented. Furthermore, a concept for integrating validation routines into the iterative model-building process is also presented.
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2222-9. doi: 10.1107/S0907444904027556. Epub 2004 Nov 26.   PUBMED

Modelling bound ligands in protein crystal structures.
Zwart PH, Langer GG, Lamzin VS
Methods for automated identification and building of protein-bound ligands in electron-density maps are described. An error model of the geometrical features of the molecular structure of a ligand based on a lattice distribution of positional parameters is obtained via simulation and is used for the construction of an approximate likelihood scoring function. This scoring function combined with a graph-based search technique provides a flexible model-building scheme and its application shows promising initial results. Several ligands with sizes ranging from 9 to 44 non-H atoms have been identified in various X-ray structures and built in an automatic way using a minimal amount of prior stereochemical knowledge.
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2230-9. doi: 10.1107/S0907444904012995. Epub 2004 Nov 26.   PUBMED

The crystal structure of glutamyl endopeptidase from Bacillus intermedius reveals a structural link between zymogen activation and charge compensation.
Meijers R, Blagova EV, Levdikov VM, Rudenskaya GN, Chestukhina GG, Akimkina TV, Kostrov SV, Lamzin VS, Kuranova IP
Extracellular glutamyl endopeptidase from Bacillus intermedius (BIEP) is a chymotrypsin-like serine protease which cleaves the peptide bond on the carboxyl side of glutamic acid. Its three-dimensional structure was determined for C222(1) and C2 crystal forms of BIEP to 1.5 and 1.75 A resolution, respectively. The topology of BIEP diverges from the most common chymotrypsin architecture, because one of the domains consists of a beta-sandwich consisting of two antiparallel beta-sheets and two helices. In the C2 crystals, a 2-methyl-2,4-pentanediol (MPD) molecule was found in the substrate binding site, mimicking a glutamic acid. This enabled the identification of the residues involved in the substrate recognition. The presence of the MPD molecule causes a change in the active site; the interaction between two catalytic residues (His47 and Ser171) is disrupted. The N-terminal end of the enzyme is involved in the formation of the substrate binding pocket. This indicates a direct relation between zymogen activation and substrate charge compensation.
Biochemistry. 2004 Mar 16;43(10):2784-91. doi: 10.1021/bi035354s.   PUBMED

The influence of positional errors on the Debye effects.
Zwart PH, Lamzin VS
The relation between a Gaussian perturbation of the atomic positional parameters and the average squared structure-factor amplitude is presented. Using an error-dependent radial distance distribution of an atomic protein model, it can be shown that the Debye effects diminish exponentially as a function of increasing positional errors. These relations can be used to estimate the quality of an atomic model and the corresponding phases. The limiting case of equal atoms with an infinitely large coordinate error results in the classical Wilson model.
Acta Crystallogr D Biol Crystallogr. 2004 Feb;60(Pt 2):220-6. doi: 10.1107/S0907444903025526. Epub 2004 Jan 23.   PUBMED


ARP/wARP and automatic interpretation of protein electron density maps.
Morris RJ, Perrakis A, Lamzin VS
no abstract available
Methods Enzymol. 2003;374:229-44. doi: 10.1016/S0076-6879(03)74011-7.   PUBMED

Distance distributions and electron-density characteristics of protein models.
Zwart PH, Lamzin VS
The analytical expression for the distribution of an interatomic distance resulting from a known error-free distance and a Gaussian perturbation of the atomic coordinates is presented. This is used to estimate the coordinate error on the basis of known geometric features of protein models via the nearest-neighbour or the radial distance distribution. A simple relation is presented that describes the dependence of the map correlation on the positional error of the protein model, the resolution of the X-ray data and the overall atomic displacement parameter. The distribution of geometrical features and the relation between the map correlation and the positional error can be used in assisting the decision-making process during automated model-building procedures.
Acta Crystallogr D Biol Crystallogr. 2003 Dec;59(Pt 12):2104-13. Epub 2003 Nov 27.   PUBMED

Breaking good resolutions with ARP/wARP.
Morris RJ, Zwart PH, Cohen S, Fernandez FJ, Kakaris M, Kirillova O, Vonrhein C, Perrakis A, Lamzin VS
New procedures are outlined that enable ARP/wARP to automatically build protein models with diffraction data extending to about 2.5 A. An overview of ongoing research is given and possible future advances are discussed.
J Synchrotron Radiat. 2004 Jan 1;11(Pt 1):56-9. Epub 2003 Nov 28.   PUBMED

Trypsin revisited: crystallography AT (SUB) atomic resolution and quantum chemistry revealing details of catalysis.
Schmidt A, Jelsch C, Ostergaard P, Rypniewski W, Lamzin VS
A series of crystal structures of trypsin, containing either an autoproteolytic cleaved peptide fragment or a covalently bound inhibitor, were determined at atomic and ultra-high resolution and subjected to ab initio quantum chemical calculations and multipole refinement. Quantum chemical calculations reproduced the observed active site crystal structure with severe deviations from standard stereochemistry and indicated the protonation state of the catalytic residues. Multipole refinement directly revealed the charge distribution in the active site and proved the validity of the ab initio calculations. The combined results confirmed the catalytic function of the active site residues and the two water molecules acting as the nucleophile and the proton donor. The crystal structures represent snapshots from the reaction pathway, close to a tetrahedral intermediate. The de-acylation of trypsin then occurs in true SN2 fashion.
J Biol Chem. 2003 Oct 31;278(44):43357-62. doi: 10.1074/jbc.M306944200. Epub 2003 Aug 22.   PUBMED

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.
Terwilliger TC, Park MS, Waldo GS, Berendzen J, Hung LW, Kim CY, Smith CV, Sacchettini JC, Bellinzoni M, Bossi R, De Rossi E, Mattevi A, Milano A, Riccardi G, Rizzi M, Roberts MM, Coker AR, Fossati G, Mascagni P, Coates AR, Wood SP, Goulding CW, Apostol MI, Anderson DH, Gill HS, Eisenberg DS, Taneja B, Mande S, Pohl E, Lamzin V, Tucker P, Wilmanns M, Colovos C, Meyer-Klaucke W, Munro AW, McLean KJ, Marshall KR, Leys D, Yang JK, Yoon HJ, Lee BI, Lee MG, Kwak JE, Han BW, Lee JY, Baek SH, Suh SW, Komen MM, Arcus VL, Baker EN, Lott JS, Jacobs W Jr, Alber T, Rupp B
The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.
Tuberculosis (Edinb). 2003;83(4):223-49.   PUBMED

Crystallization and preliminary X-ray analysis of a four-copper laccase from Coriolus hirsutus.
Pegasova TV, Zwart P, Koroleva OV, Stepanova EV, Rebrikov DV, Lamzin VS
Laccase from the fungus Coriolus hirsutus has been purified. Crystals of the enzyme suitable for X-ray structure analysis have been obtained under optimized crystallization conditions using polyethylene glycol as a precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.65, b = 74.01, c = 124.83 A, and contain 40% solvent and a single molecule of laccase in the asymmetric unit. X-ray data were collected to 1.85 A at the copper edge and the four copper sites have been located from the anomalous signal. The obtained SAD phases with subsequent density modification produced a promising initial electron-density map.
Acta Crystallogr D Biol Crystallogr. 2003 Aug;59(Pt 8):1459-61. Epub 2003 Jul 23.   PUBMED


Veni, vidi, vici - atomic resolution unravelling the mysteries of protein function.
Schmidt A, Lamzin VS
Atomic resolution macromolecular crystallography has become a powerful and versatile tool in structural biology; the number of atomic resolution structures is steadily increasing. Novel techniques are being developed and the use of complementary methods that span the field from sample preparation to validation and analysis of the resulting models has emerged. These allow the fuller exploitation of the information stored in crystal structures and reveal a depth of structural detail that was unattainable in the recent past.
Curr Opin Struct Biol. 2002 Dec;12(6):698-703.   PUBMED

Advantages of high-resolution phasing: MAD to atomic resolution.
Schmidt A, Gonzalez A, Morris RJ, Costabel M, Alzari PM, Lamzin VS
The structure of the endoglucanase A from Clostridium thermocellum CelA was re-solved by three-wavelength MAD. Experimental phases were obtained in the resolution range 25-1.0 A. Various structure-solution approaches were tested in order to quantify the contribution of each wavelength. Two-wavelength MAD phasing was sufficient to obtain excellent experimental phases. SAD at the remote wavelength also resulted in interpretable maps. The three-wavelength MAD electron-density map was of excellent quality: for parts of the structure, atom types and bond types could be easily assigned. Double bonds in peptide links and side chains could be located owing to their increased electron density indicating their pi character. Comparison with a previously determined structure of CelA at 1.65 A showed that, apart from a few additional multiple conformers and differently oriented side chains, major differences occur at the protein-solvent interface. A complete additional solvent shell could be observed and the inner shells have been completed. The high accuracy of the structure allowed unambiguous assignment of the protonation state for the active-site catalytic carboxylates.
Acta Crystallogr D Biol Crystallogr. 2002 Sep;58(Pt 9):1433-41. doi: 10.1107/S0907444902011368. Epub 2002 Aug 23.   PUBMED

Atomic resolution data reveal flexibility in the structure of RNase Sa.
Sevcik J, Lamzin VS, Dauter Z, Wilson KS
Ribonuclease from Streptomyces aureofaciens, the bacterial source for the industrial production of chlorotetracycline, is a guanylate endoribonuclease (RNase Sa; EC which hydrolyses the phosphodiester bonds of single-stranded RNA at the 3'-side of guanosine nucleotides with high specificity. The structure of the enzyme was previously refined at atomic resolution (1.2 A) using room-temperature data. Here, the RNase Sa structure refined against 1.0 A data collected at cryogenic temperature is reported. There are two surface loops in molecule A and one in molecule B for which two main-chain conformations are modelled: these loops contain active-site residues. The separation for most of the corresponding main-chain atoms in the two conformations is about 0.8 A, with a maximum of 2.5 A. The two regions of dual conformation represent the most important differences in comparison with the structure determined at room temperature, where the corresponding loops have one conformation only but the largest degree of anisotropy. The flexibility of the loops observed in the structure of RNase Sa is directly linked to the need for the active site to interact productively with substrates and/or inhibitors.
Acta Crystallogr D Biol Crystallogr. 2002 Aug;58(Pt 8):1307-13. Epub 2002 Jul 20.   PUBMED

Domain closure, substrate specificity and catalysis of D-lactate dehydrogenase from Lactobacillus bulgaricus.
Razeto A, Kochhar S, Hottinger H, Dauter M, Wilson KS, Lamzin VS
NAD-dependent Lactobacillus bulgaricus D-Lactate dehydrogenase (D-LDHb) catalyses the reversible conversion of pyruvate into D-lactate. Crystals of D-LDHb complexed with NADH were grown and X-ray data collected to 2.2 A. The structure of D-LDHb was solved by molecular replacement using the dimeric Lactobacillus helveticus D-LDH as a model and was refined to an R-factor of 20.7%. The two subunits of the enzyme display strong asymmetry due to different crystal environments. The opening angles of the two catalytic domains with respect to the core coenzyme binding domains differ by 16 degrees. Subunit A is in an "open" conformation typical for a dehydrogenase apo enzyme and subunit B is "closed". The NADH-binding site in subunit A is only 30% occupied, while in subunit B it is fully occupied and there is a sulphate ion in the substrate-binding pocket. A pyruvate molecule has been modelled in the active site and its orientation is in agreement with existing kinetic and structural data. On domain closure, a cluster of hydrophobic residues packs tightly around the methyl group of the modelled pyruvate molecule. At least three residues from this cluster govern the substrate specificity. Substrate binding itself contributes to the stabilisation of domain closure and activation of the enzyme. In pyruvate reduction, D-LDH can adapt another protonated residue, a lysine residue, to accomplish the role of the acid catalyst His296. Required lowering of the lysine pK(a) value is explained on the basis of the H296K mutant structure.
J Mol Biol. 2002 Apr 19;318(1):109-19. doi: 10.1016/S0022-2836(02)00086-4.   PUBMED

ARP/wARP's model-building algorithms. I. The main chain.
Morris RJ, Perrakis A, Lamzin VS
Algorithms underlying the automatic model-building functionality of the ARP/wARP software suite are presented. Finding the most likely set of Calpha atoms from a given set of atoms is formulated as a constrained integer programming problem. The objective function is a density-weighted score for the match between observed and expected chain conformation. Graph-search algorithms are presented that find solutions to this problem in an efficient manner.
Acta Crystallogr D Biol Crystallogr. 2002 Jun;58(Pt 6 Pt 2):968-75. Epub 2002 May 29.   PUBMED

Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate.
Guerin DM, Lascombe MB, Costabel M, Souchon H, Lamzin V, Beguin P, Alzari PM
The crystal structure of Clostridium thermocellum endoglucanase CelA in complex with cellopentaose has been determined at 0.94 A resolution. The oligosaccharide occupies six D-glucosyl-binding subsites, three on either side of the scissile glycosidic linkage. The substrate and product of the reaction occupy different positions at the reducing end of the cleft, where an extended array of hydrogen-bonding interactions with water molecules fosters the departure of the leaving group. Severe torsional strain upon the bound substrate forces a distorted boat(2,5) B conformation for the glucosyl residue bound at subsite -1, which facilitates the formation of an oxocarbenium ion intermediate and might favor the breakage of the sugar ring concomitant with catalysis.
J Mol Biol. 2002 Mar 8;316(5):1061-9. doi: 10.1006/jmbi.2001.5404.   PUBMED

Atomic resolution structures of ribonuclease A at six pH values.
Berisio R, Sica F, Lamzin VS, Wilson KS, Zagari A, Mazzarella L
The diffraction pattern of protein crystals extending to atomic resolution guarantees a very accurate picture of the molecular structure and enables the study of subtle phenomena related to protein functionality. Six structures of bovine pancreatic ribonuclease at the pH* values 5.2, 5.9, 6.3, 7.1, 8.0 and 8.8 and at resolution limits in the range 1.05-1.15A have been refined. An overall description of the six structures and several aspects, mainly regarding pH-triggered conformational changes, are described here. Since subtle variations were expected, a thorough validation assessment of the six refined models was first carried out. Some stereochemical parameters, such as the N[bond]C(alpha)[bond]C angle and the pyramidalization at the carbonyl C atoms, indicate that the standard target values and their weights typically used in refinement may need revision. A detailed comparison of the six structures has provided experimental evidence on the role of Lys41 in catalysis. Furthermore, insights are given into the structural effects related to the pH-dependent binding of a sulfate anion, which mimics the phosphate group of RNA, in the active site. Finally, the results support a number of thermodynamic and kinetic experimental data concerning the role of the disulfide bridge between Cys65 and Cys72 in the folding of RNase A.
Acta Crystallogr D Biol Crystallogr. 2002 Mar;58(Pt 3):441-50. Epub 2002 Feb 21.   PUBMED

The structures of Escherichia coli inorganic pyrophosphatase complexed with Ca(2+) or CaPP(i) at atomic resolution and their mechanistic implications.
Samygina VR, Popov AN, Rodina EV, Vorobyeva NN, Lamzin VS, Polyakov KM, Kurilova SA, Nazarova TI, Avaeva SM
Two structures of Escherichia coli soluble inorganic pyrophosphatase (EPPase) complexed with calcium pyrophosphate (CaPP(i)-EPPase) and with Ca(2+) (Ca(2+)-EPPase) have been solved at 1.2 and 1.1 A resolution, respectively. In the presence of Mg(2+), this enzyme cleaves pyrophosphate (PP(i)) into two molecules of orthophosphate (P(i)). This work has enabled us to locate PP(i) in the active site of the inorganic pyrophosphatases family in the presence of Ca(2+), which is an inhibitor of EPPase.Upon PP(i) binding, two Ca(2+) at M1 and M2 subsites move closer together and one of the liganded water molecules becomes bridging. The mutual location of PP(i) and the bridging water molecule in the presence of inhibitor cation is catalytically incompetent. To make a favourable PP(i) attack by this water molecule, modelling of a possible hydrolysable conformation of PP(i) in the CaPP(i)-EPPase active site has been performed. The reasons for Ca(2+) being the strong PPase inhibitor and the role in catalysis of each of four metal ions are the mechanistic aspects discussed on the basis of the structures described.
J Mol Biol. 2001 Nov 30;314(3):633-45. doi: 10.1006/jmbi.2001.5149.   PUBMED

Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre.
Galkin AG, Kutsenko AS, Bajulina NP, Esipova NG, Lamzin VS, Mesentsev AV, Shelukho DV, Tikhonova TV, Tishkov VI, Ustinnikova TB, Popov VO
Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp. 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases. Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH. In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds. In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain. The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity. CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated. It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure. The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding. In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.
Biochim Biophys Acta. 2002 Jan 31;1594(1):136-49.   PUBMED


Crystal structure of manganese catalase from Lactobacillus plantarum.
Barynin VV, Whittaker MM, Antonyuk SV, Lamzin VS, Harrison PM, Artymiuk PJ, Whittaker JW
BACKGROUND: Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex. RESULTS: The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase. CONCLUSIONS: A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.
Structure. 2001 Aug;9(8):725-38.   PUBMED

ARP/wARP and molecular replacement.
Perrakis A, Harkiolaki M, Wilson KS, Lamzin VS
The aim of ARP/wARP is improved automation of model building and refinement in macromolecular crystallography. Once a molecular-replacement solution has been obtained, it is often tedious to refine and rebuild the initial (search) model. ARP/wARP offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an improved map and (iii) docking of a structure onto a new (or mutated) sequence, followed by rebuilding and refining the side chains in real space. A few examples are presented where ARP/wARP made a considerable difference in the speed of structure solution and/or made possible refinement of otherwise difficult or uninterpretable maps. The resolution range allowing complete autobuilding of protein structures is currently 2.0 A, but for map improvement considerable advances over more conventional refinement techniques are evident even at 3.2 A spacing.
Acta Crystallogr D Biol Crystallogr. 2001 Oct;57(Pt 10):1445-50. Epub 2001 Sep 21.   PUBMED

Protein crystal growth in the Advanced Protein Crystallization Facility on the LMS mission: a comparison of Sulfolobus solfataricus alcohol dehydrogenase crystals grown on the ground and in microgravity.
Esposito L, Sica F, Sorrentino G, Berisio R, Carotenuto L, Giordano A, Raia CA, Rossi M, Lamzin VS, Wilson KS, Zagari A
Crystals of alcohol dehydrogenase from Sulfolobus solfataricus were grown in the Advanced Protein Crystallization Facility during the Life and Microgravity Sciences Spacelab mission on the US Space Shuttle. Large diffracting crystals were obtained by dialysis, whereas only poor-quality crystals were obtained by vapour diffusion. The quality of both the microgravity and ground-based crystals was analysed by X-ray diffraction. There was some improvement in terms of size and diffraction resolution limit for the microgravity crystals. However, the twinning observed in the Earth-grown crystals was also present for those grown in microgravity.
Acta Crystallogr D Biol Crystallogr. 1998 May 1;54(Pt 3):386-90.   PUBMED

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding.
Grabarse W, Mahlert F, Duin EC, Goubeaud M, Shima S, Thauer RK, Lamzin V, Ermler U
Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.
J Mol Biol. 2001 May 25;309(1):315-30. doi: 10.1006/jmbi.2001.4647.   PUBMED

Detailed analysis of RNA-protein interactions within the ribosomal protein S8-rRNA complex from the archaeon Methanococcus jannaschii.
Tishchenko S, Nikulin A, Fomenkova N, Nevskaya N, Nikonov O, Dumas P, Moine H, Ehresmann B, Ehresmann C, Piendl W, Lamzin V, Garber M, Nikonov S
The crystal structure of ribosomal protein S8 bound to its target 16 S rRNA from a hyperthermophilic archaeon Methanococcus jannaschii has been determined at 2.6 A resolution. The protein interacts with the minor groove of helix H21 at two sites located one helical turn apart, with S8 forming a bridge over the RNA major groove. The specificity of binding is essentially provided by the C-terminal domain of S8 and the highly conserved nucleotide core, characterized by two dinucleotide platforms, facing each other. The first platform (A595-A596), which is the less phylogenetically and structurally constrained, does not directly contact the protein but has an important shaping role in inducing cross-strand stacking interactions. The second platform (U641-A642) is specifically recognized by the protein. The universally conserved A642 plays a pivotal role by ensuring the cohesion of the complex organization of the core through an array of hydrogen bonds, including the G597-C643-U641 base triple. In addition, A642 provides the unique base-specific interaction with the conserved Ser105, while the Thr106 - Thr107 peptide link is stacked on its purine ring. Noteworthy, the specific recognition of this tripeptide (Thr-Ser-Thr/Ser) is parallel to the recognition of an RNA tetraloop by a dinucleotide platform in the P4-P6 ribozyme domain of group I intron. This suggests a general dual role of dinucleotide platforms in recognition of RNA or peptide motifs. One prominent feature is that conserved side-chain amino acids, as well as conserved bases, are essentially involved in maintaining tertiary folds. The specificity of binding is mainly driven by shape complementarity, which is increased by the hydrophobic part of side-chains. The remarkable similarity of this complex with its homologue in the T. thermophilus 30 S subunit indicates a conserved interaction mode between Archaea and Bacteria.
J Mol Biol. 2001 Aug 10;311(2):311-24. doi: 10.1006/jmbi.2001.4877.   PUBMED

Non-covalent interactions in the crystallization of the enantiomers of 1,7-dioxaspiro.
Makedonopoulou S, Yannakopoulou K, Mentzafos D, Lamzin V, Popov A, Mavridis IM
The enantiomers of racemic olive fly sex pheromone 1,7-dioxaspiro[5.5]undecane (1) have been isolated by crystallization with enantiospecific cyclodextrin hosts: (S)-(1) crystallizes with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) and (R)-(1) with hexakis(2,3,6-tri-O-methyl)-alpha-cyclodextrin (TMalphaCD). The crystal structure of TMbetaCD/(S)-(1) from synchrotron radiation data at 100 K, determined for the first time, proves that TMbetaCD crystallizes with only the (S)-enantiomer from the racemic mixture. Comparison with the 100 K structure of TMalphaCD/(R)-(1) redetermined with synchrotron data has provided insight into the interactions between each of the hosts with the corresponding enantiomeric guests. Owing to the high resolution of the data and the unusually high quality of the crystals, localization of the H atoms has been achieved, a rare accomplishment for cyclodextrin X-ray structures. This made possible, apart from the geometry of the complexes, the detailed description of a five-membered-ring water cluster with very well ordered hydrogen bonding. The enantiospecificity exhibited by the described systems reveals the subtle differences of the weak intermolecular forces involved in the selective binding of the two optical antipodes by the two hosts. The binding geometry in the two complexes is different, but it is effected in both by numerous host-guest C-H.O interactions, resulting from induced fit of the hosts toward each of the enantiomeric guests. In TMalphaCD/(R)-(1) two of these H.O host-guest distances, directed toward the acetal O atoms defining the chirality of the guest, are much shorter than the rest and also shorter than all the H.O distances in TMbetaCD/(S)-(1). Moreover, (R)-(1) interacts not only with the enclosing host, but with other hosts in the crystal lattice, in contrast to (S)-(1) in the TMbetaCD/(S)-(1) complex which is isolated inside channels formed by the host molecules. The above differences are reflected in the much higher binding constant of TMalphaCD/(R)-(1) compared with that of TMbetaCD/(S)-(1) ( approximately 6800 and approximately 935 M(-1), respectively), determined by NMR in aqueous solution, and the ability of TMalphaCD to selectively precipitate (R)-(1) from racemic (1) in much higher yield than TMbetaCD precipitates (S)-(1).
Acta Crystallogr B. 2001 Jun;57(Pt 3):399-409. Epub 2001 Jun 1.   PUBMED

Transthyretin stability as a key factor in amyloidogenesis: X-ray analysis at atomic resolution.
Sebastiao MP, Lamzin V, Saraiva MJ, Damas AM
Transthyretin (TTR) amyloidosis is a conformational disturbance, which, like other amyloidoses, represents a life threat. Here, we report a TTR variant, TTR Thr119Met, that has been shown to have a protective role in the development of clinical symptoms in carriers of TTR Val30Met, one of the most frequent variants among TTR amyloidosis patients. In order to understand this effect, we have determined the structures of the TTR Val30Met/Thr119Met double mutant isolated from the serum of one patient and of both the native and thyroxine complex of TTR Thr119Met. Major conclusions are: (i) new H-bonds within each monomer and monomer-monomer inter-subunit contacts, e.g. Ser117-Ser117 and Met119-Tyr114, increase protein stability, possibly leading to the protective effect of the TTR Val30Met/Thr119Met variant when compared to the single variant TTR Val30Met. (ii) The mutated residue (Met119) extends across the thyroxine binding channel inducing conformational changes that lead to closer contacts between different dimers within the tetramer. The data, at atomic resolution, were essential to detect, for the first time, the subtle changes in the inter-subunit contacts of TTR, and explain the non-amyloidogenic potential of the TTR Thr119Met variant, improving considerably current research on the TTR amyloid fibril formation pathway.
J Mol Biol. 2001 Mar 2;306(4):733-44. doi: 10.1006/jmbi.2000.4415.   PUBMED

On the enzymatic activation of NADH.
Meijers R, Morris RJ, Adolph HW, Merli A, Lamzin VS, Cedergren-Zeppezauer ES
Atomic (1 A) resolution x-ray structures of horse liver alcohol dehydrogenase in complex with NADH revealed the formation of an adduct in the active site between a metal-bound water and NADH. Furthermore, a pronounced distortion of the pyridine ring of NADH was observed. A series of quantum chemical calculations on the water-nicotinamide adduct showed that the puckering of the pyridine ring in the crystal structures can only be reproduced when the water is considered a hydroxide ion. These observations provide fundamental insight into the enzymatic activation of NADH for hydride transfer.
J Biol Chem. 2001 Mar 23;276(12):9316-21. doi: 10.1074/jbc.M010870200. Epub 2000 Dec 28.   PUBMED


Current state of automated crystallographic data analysis.
Lamzin VS, Perrakis A
A goal of structural biology--and of structural genomics in particular--is to improve the underlying methodology for high-throughput determination of three-dimensional structures of biological macromolecules. Here we address issues related to the development, automation and streamlining of the process of macromolecular X-ray crystal structure solution.
Nat Struct Biol. 2000 Nov;7 Suppl:978-81. doi: 10.1038/80763.   PUBMED

Apotheosis, not apocalypse: methods in protein crystallography.
Lamzin VS, Perrakis A, Bricogne G, Jiang J, Swaminathan S, Sussman JL
no abstract available
Acta Crystallogr D Biol Crystallogr. 2000 Nov;56(Pt 11):1510-1.   PUBMED

Structural basis for substrate specificity differences of horse liver alcohol dehydrogenase isozymes.
Adolph HW, Zwart P, Meijers R, Hubatsch I, Kiefer M, Lamzin V, Cedergren-Zeppezauer E
A structure determination in combination with a kinetic study of the steroid converting isozyme of horse liver alcohol dehydrogenase, SS-ADH, is presented. Kinetic parameters for the substrates, 5beta-androstane-3beta,17beta-ol, 5beta-androstane-17beta-ol-3-one, ethanol, and various secondary alcohols and the corresponding ketones are compared for the SS- and EE-isozymes which differ by nine amino acid substitutions and one deletion. Differences in substrate specificity and stereoselectivity are explained on the basis of individual kinetic rate constants for the underlying ordered bi-bi mechanism. SS-ADH was crystallized in complex with 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan -24-acid (cholic acid) and NAD(+), but microspectrophotometric analysis of single crystals proved it to be a mixed complex containing 60-70% NAD(+) and 30-40% NADH. The crystals belong to the space group P2(1) with cell dimensions a = 55.0 A, b = 73.2 A, c = 92.5 A, and beta = 102.5 degrees. A 98% complete data set to 1.54-A resolution was collected at 100 K using synchrotron radiation. The structure was solved by the molecular replacement method utilizing EE-ADH as the search model. The major structural difference between the isozymes is a widening of the substrate channel. The largest shifts in C(alpha) carbon positions (about 5 A) are observed in the loop region, in which a deletion of Asp115 is found in the SS isozyme. SS-ADH easily accommodates cholic acid, whereas steroid substrates of similar bulkiness would not fit into the EE-ADH substrate site. In the ternary complex with NAD(+)/NADH, we find that the carboxyl group of cholic acid ligates to the active site zinc ion, which probably contributes to the strong binding in the ternary NAD(+) complex.
Biochemistry. 2000 Oct 24;39(42):12885-97.   PUBMED

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase.
Samygina VR, Antonyuk SV, Lamzin VS, Popov AN
A significant improvement in the X-ray resolution of crystals of Escherichia coli inorganic pyrophosphatase at cryotemperature was obtained as a result of studying the relationship between the crystal order and cryosolution component concentrations. To perform the experiments, the ability to reverse the flash-cooling process and to return a crystal to ambient temperature was used. In each cycle, the crystal was transferred from a cold nitrogen-gas stream to a cryosolution with modified concentrations of the components. The crystal was then flash-cooled again and the diffraction quality checked. Such a technique allows the screening of a wide concentration range rather quickly without using a large number of crystals and allows the determination of optimal cryosolution component concentrations. The resolution limit for crystals of pyrophosphatase increased by almost 0.7 A, from 1.8 to 1.15 A.
Acta Crystallogr D Biol Crystallogr. 2000 May;56(Pt 5):595-603.   PUBMED

Accurate protein crystallography at ultra-high resolution: valence electron distribution in crambin.
Jelsch C, Teeter MM, Lamzin V, Pichon-Pesme V, Blessing RH, Lecomte C
The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 A) at low temperature by using short-wavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.
Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3171-6.   PUBMED

Roles of his205, his296, his303 and Asp259 in catalysis by NAD+-specific D-lactate dehydrogenase.
Kochhar S, Lamzin VS, Razeto A, Delley M, Hottinger H, Germond JE
The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.
Eur J Biochem. 2000 Mar;267(6):1633-9.   PUBMED


Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.
Declercq JP, Evrard C, Lamzin V, Parello J
Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.
Protein Sci. 1999 Oct;8(10):2194-204. doi: 10.1110/ps.8.10.2194.   PUBMED

Ab initio solution and refinement of two high-potential iron protein structures at atomic resolution.
Parisini E, Capozzi F, Lubini P, Lamzin V, Luchinat C, Sheldrick GM
The crystal structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0. 93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R(1) = summation operator||F(o)| - |F(c)|| / summation operator|F(o)| of 12.0 and 13.6%, respectively [data with I > 2sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe(4)S(4)](2+) cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe-S bonds along four approximately parallel cube edges, and eight long Fe-S bonds. The unique protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the protein rather than by structural differences in the cluster region.
Acta Crystallogr D Biol Crystallogr. 1999 Nov;55(Pt 11):1773-84.   PUBMED

Protein titration in the crystal state.
Berisio R, Lamzin VS, Sica F, Wilson KS, Zagari A, Mazzarella L
Proteins are complex structures whose overall stability critically depends on a delicate balance of numerous interactions of similar strength, which are markedly influenced by their environment. Here, we present an analysis of the effect of pH on a protein structure in the crystalline state using RNase A as a model system. By altering only one physico-chemical parameter in a controlled manner, we are able to quantify the structural changes induced in the protein. Atomic resolution X-ray diffraction data were collected for crystals at six pH* values ranging from 5.2 to 8.8, and the six independently refined structures reveal subtle, albeit well-defined variations directly related to the pH titration of the protein. The deprotonation of the catalytic His12 residue is clearly evident in the electron density maps, confirming the reaction mechanism proposed by earlier enzymatic and structural studies. The concerted structural changes observed in the regions remote from the active-site point to an adaptation of the protein structure to the changes in the physico-chemical environment. Analysis of the stereochemistry of the six structures provided accurate estimates of p Kavalues of most of the histidine residues. This study gives further evidence for the advantage of atomic resolution X-ray crystallographic analyses for revealing small but significant structural changes which provide clues to the function of a biological macromolecule.
J Mol Biol. 1999 Oct 1;292(4):845-54. doi: 10.1006/jmbi.1999.3093.   PUBMED

Ab initio structure solution of a dimeric cytochrome c3 from Desulfovibrio gigas containing disulfide bridges.
Frazao C, Sieker L, Sheldrick G, Lamzin V, LeGall J, Carrondo MA
The 1.2 A resolution crystal structure of the 29 kDa di-tetrahaem cytochrome c3 from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c3 from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c3 molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.
J Biol Inorg Chem. 1999 Apr;4(2):162-5.   PUBMED

Molecular, crystal and solution structure of a beta-cyclodextrin complex with the bromide salt of 2-(3-dimethylaminopropyl)tricyclo[,7)]decan-2-ol, a potent antimicrobial drug.
Perrakis A, Antoniadou-Vyza E, Tsitsa P, Lamzin VS, Wilson KS, Hamodrakas SJ
The pharmacological properties of a cyclomaltoheptaose (beta-cyclodextrin) series of adamantane-group-bearing compounds that exhibit potent antibacterial activity have been studied, both isolated and in complex with beta-cyclodextrins (betaCDs). In this work, the structure of the bromide salt of 2-(3-dimethylaminopropyl)-tricyclo[,7)]decan-2-ol(A DM-10) complexed with betaCD and ten water molecules was studied in the solid state by X-ray crystallography and in solution by NMR spectroscopy. X-ray crystallographic studies of the complex were performed both at room and cryogenic temperatures. The long aliphatic chain of ADM-10 adopts a single conformation at low temperature in contrast to what is observed at room temperature, where two side chain conformations are seen. Both NMR and X-ray crystallography studies indicate that the adamantane moiety of ADM-10 is buried in the betaCD cavity. Chemical shifts in NMR experiments can be explained on the basis of the crystal structure of the complex.
Carbohydr Res. 1999 Apr 30;317(1-4):19-28.   PUBMED

Experimental observation of bonding electrons in proteins.
Lamzin VS, Morris RJ, Dauter Z, Wilson KS, Teeter MM
We demonstrate with two examples the success and potential of recent developments in x-ray protein crystallography at ultra high resolution. Our preliminary structural analyses using diffraction data collected for the two proteins crambin and savinase show meaningful deviations from the conventional independent spherical atom approximation. A noise-reduction averaging technique enables bonding details of electron distributions in proteins to be revealed experimentally for the first time. We move one step closer to imaging directly the fine details of the electronic structure on which the biological function of a protein is based.
J Biol Chem. 1999 Jul 23;274(30):20753-5.   PUBMED

Automated protein model building combined with iterative structure refinement.
Perrakis A, Morris R, Lamzin VS
In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.
Nat Struct Biol. 1999 May;6(5):458-63. doi: 10.1038/8263.   PUBMED


Accelerated X-ray structure elucidation of a 36 kDa muramidase/transglycosylase using wARP.
Van Asselt EJ, Perrakis A, Kalk KH, Lamzin VS, Dijkstra BW
The X-ray structure of the 36 kDa soluble lytic transglycosylase from Escherichia coli has been determined starting with the multiple isomorphous replacement method with inclusion of anomalous scattering at 2.7 A resolution. Subsequently, before any model building was carried out, phases were extended to 1.7 A resolution with the weighted automated refinement procedure wARP, which gave a dramatic improvement in the phases. The electron-density maps from wARP were of outstanding quality for both the main chain and the side chains of the protein, which allowed the time spent on the tracing, interpretation and building of the X-ray structure to be substantially shortened. The structure of the soluble lytic transglycosylase was refined at 1.7 A resolution with X-PLOR to a final crystallographic R factor of 18.9%. Analysis of the wARP procedure revealed that the use of the maximum-likelihood refinement in wARP gave much better phases than least-squares refinement, provided that the ratio of reflections to protein atom parameters was approximately 1.8 or higher. Furthermore, setting aside 5% of the data for an Rfree test set had a negative effect on the phase improvement. The mean WwARP, a weight determined at the end of the wARP procedure and based on the variance of structure factors from six individually refined wARP models, proved to be a better indicator than the Rfree factor to judge different phase improvement protocols. The elongated Slt35 structure has three domains named the alpha, beta and core domains. The alpha domain contains mainly alpha-helices, while the beta domain consists of a five-stranded antiparallel beta-sheet flanked by a short alpha-helix. Sandwiched between the alpha and beta domains is the core domain, which bears some resemblance to the fold of the catalytic domain of the previously elucidated 70 kDa soluble lytic transglycosylase from E. coli. The putative active site is at the bottom of a large deep groove in the core domain.
Acta Crystallogr D Biol Crystallogr. 1998 Jan 1;54(Pt 1):58-73.   PUBMED

Refinement of triclinic hen egg-white lysozyme at atomic resolution.
Walsh MA, Schneider TR, Sieker LC, Dauter Z, Lamzin VS, Wilson KS
X-ray diffraction data have been collected at both low (120 K) and room temperature from triclinic crystals of hen egg-white lysozyme to 0.925 and 0.950 A resolution, respectively, using synchrotron radiation. Data from one crystal were sufficient for the low-temperature study, whereas three crystals were required at room temperature. Refinement was carried out using the programs PROLSQ, ARP and SHELXL to give final conventional R factors of 8.98 and 10.48% for data with F > 4sigma(F) for the low- and room-temperature structures, respectively. The estimated r.m.s. coordinate error is 0.032 A for protein atoms, 0.050 A for all atoms in the low-temperature study, and 0.038 A for protein atoms and 0.049 A for all atoms in the room-temperature case, as estimated from inversion of the blocked least-squares matrix. The low-temperature study revealed that the side chains of 24 amino acids had multiple conformations. A total of 250 waters, six nitrate ions and three acetate ions, two of which were modelled with alternate orientations were located in the electron-density maps. Three sections of the main chain were modelled in alternate conformations. The room-temperature study produced a model with multiple conformations for eight side chains and a total of 139 water molecules, six nitrate but no acetate ions. The occupancies of the water molecules were refined in both structures and this step was shown to be meaningful when assessed by use of the free R factor. A detailed description and comparison of the structures is made with reference to the previously reported structure refined at 2.0 A resolution.
Acta Crystallogr D Biol Crystallogr. 1998 Jul 1;54(Pt 4):522-46.   PUBMED

Validation tools: can they indicate the information content of macromolecular crystal structures?
Dodson EJ, Davies GJ, Lamzin VS, Murshudov GN, Wilson KS
The explosive increase in the number of published three-dimensionsal structures of macromolecules determined by X-ray analysis places a responsibility on experimentalists, referees and curators of databases to ensure correspondence between the structure parameters and data. Validation tools will evolve as more appropriate statistical techniques and new information, such as that from proteins analysed at atomic resolution, becomes available.
Structure. 1998 Jun 15;6(6):685-90. doi: 10.1016/S0969-2126(98)00070-7.   PUBMED

The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.
Levdikov VM, Barynin VV, Grebenko AI, Melik-Adamyan WR, Lamzin VS, Wilson KS
BACKGROUND: The biosynthesis of key metabolic components is of major interest to biologists. Studies of de novo purine synthesis are aimed at obtaining a deeper understanding of this central pathway and the development of effective chemotherapeutic agents. Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase catalyses the seventh step out of ten in the biosynthesis of purine nucleotides. To date, only one structure of an enzyme involved in purine biosynthesis has been reported: adenylosuccinate synthetase, which catalyses the first committed step in the synthesis of AMP from IMP. RESULTS: We report the first three-dimensional structure of a SAICAR synthase, from Saccharomyces cerevisiae. It is a monomer with three domains. The first two domains consist of antiparallel beta sheets and the third is composed of two alpha helices. There is a long deep cleft made up of residues from all three domains. Comparison of SAICAR synthases by alignment of their sequences reveals a number of conserved residues, mostly located in the cleft. The presence of two sulphate ions bound in the cleft, the structure of SAICAR synthase in complex with ATP and a comparison of this structure with that of other ATP-dependent proteins point to the interdomain cleft as the location of the active site. CONCLUSIONS: The topology of the first domain of SAICAR synthase resembles that of the N-terminal domain of proteins belonging to the cyclic AMP-dependent protein kinase family. The fold of the second domain is similar to that of members of the D-alanine:D-alanine ligase family. Together these enzymes form a new superfamily of mononucleotide-binding domains. There appears to be no other enzyme, however, which is composed of the same combination of three domains, with the individual topologies found in SAICAR synthase.
Structure. 1998 Mar 15;6(3):363-76.   PUBMED

Conserved supersecondary structural motif in NAD-dependent dehydrogenases.
Kutzenko AS, Lamzin VS, Popov VO
L- and D-specific nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenases map to the same structural protein superfamily as defined by the Structural Classification of Proteins (SCOP) and are based on the Rossmann fold type domains. A detailed classification of these domains is proposed using a novel diagnostic parameter of the rms per aligned pair. The catalytic domain in D-specific dehydrogenases shows a strong structural homology to the coenzyme binding domain. A topologically conserved part within the dehydrogenase superfamily reveals a supersecondary structural motif comprising the 5-stranded left-handedly twisted parallel beta-sheet with one complete and one partial Rossmann fold units and two alpha-helices, the long helix, adjacent to and running roughly parallel with the beta-sheet plane and the helix connecting two Rossmann folds.
FEBS Lett. 1998 Feb 13;423(1):105-9.   PUBMED


The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism.
Mathieu M, Modis Y, Zeelen JP, Engel CK, Abagyan RA, Ahlberg A, Rasmussen B, Lamzin VS, Kunau WH, Wierenga RK
The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms. This reaction is the last step of the beta-oxidation pathway. The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution. An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets. The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices. The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules. The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket. The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism. From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues. In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior. The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A. The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction. The CoA moiety is proposed to bind in a groove on the surface of the protein molecule. Most of the interactions of the CoA molecule are with atoms of the loop domain. The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases.
J Mol Biol. 1997 Oct 31;273(3):714-28. doi: 10.1006/jmbi.1997.1331.   PUBMED

The benefits of atomic resolution.
Dauter Z, Lamzin VS, Wilson KS
After a long gestation, the elucidation of the crystal structures of proteins at atomic resolution is now maturing. The use of such data for both refinement and structure solution is advancing space. The necessary technology is generally available, in terms of data collection, computing hardware and software. The structures appearing in the literature mainly relate to demonstration projects on native proteins. The importance of these alone is already obvious. Biologically significant results, in terms of ligand complexes and prosthetic groups, are just starting to emerge.
Curr Opin Struct Biol. 1997 Oct;7(5):681-8.   PUBMED

wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models.
Perrakis A, Sixma TK, Wilson KS, Lamzin VS
wARP is a procedure that substantially improves crystallographic phases (and subsequently electron-density maps) as an additional step after density-modification methods such as solvent flattening and averaging. The initial phase set is used to create a number of dummy atom models which are subjected to least-squares or maximum-likelihood refinement and iterative model updating in an automated refinement procedure (ARP). Averaging of the phase sets calculated from the refined output models and weighting of structure factors by their similarity to an average vector results in a phase set that improves and extends the initial phases substantially. An important requirement is that the native data have a maximum resolution beyond approximately 2.4 A. The wARP procedure shortens the time-consuming step of model building in crystallographic structure determination and helps to prevent the introduction of errors.
Acta Crystallogr D Biol Crystallogr. 1997 Jul 1;53(Pt 4):448-55. doi: 10.1107/S0907444997005696.   PUBMED

Atomic resolution (1.0 A) crystal structure of Fusarium solani cutinase: stereochemical analysis.
Longhi S, Czjzek M, Lamzin V, Nicolas A, Cambillau C
X-ray data have been recorded to 1.0 A resolution from a crystal of Fusarium solani cutinase using synchrotron radiation and an imaging-plate scanner. The anisotropic treatment of thermal motion led to a fivefold increase in accuracy and to a considerable quality improvement in the electron density maps with respect to an intermediate isotropic model. The final model has an R-factor of 9.4%, with a mean coordinate error of 0.021 A, as estimated from inversion of the least-squares matrix. The availability of an accurate structure at atomic resolution and of meaningful estimates of the errors in its atomic parameters, allowed an extensive analysis of several stereochemical parameters, such as peptide planarity, main-chain and some side-chain bond distances. The hydrogen atoms could be clearly identified in the electron density, thus providing unambiguous evidence on the protonation state of the catalytic histidine residue. The atomic resolution revealed an appreciable extent of flexibility in the cutinase active site, which might be correlated with a possible adaptation to different substrates. The anisotropic treatment of thermal factors provided insights into the anisotropic nature of motions. The analysis of these motions in the two loops delimiting the catalytic crevice pointed out a "breath-like" movement in the substrate binding region of cutinase.
J Mol Biol. 1997 May 16;268(4):779-99. doi: 10.1006/jmbi.1997.1000.   PUBMED

Effect of pH on kinetic parameters of NAD+-dependent formate dehydrogenase.
Mesentsev AV, Lamzin VS, Tishkov VI, Ustinnikova TB, Popov VO
To define in detail the molecular mechanism of NAD+-dependent formate dehydrogenase, the pH dependences of various kinetic and spectroscopic parameters have been studied: Vmax, Km (NAD+), Km (formate), inhibition constants for structural analogues of substrate (NO3-) and product (CNS-, CNO-, N3-), CD and fluorescence properties. The value of Vmax, rate-limiting hydride transfer, is nearly constant throughout the entire pH range of enzyme stability (6.0-11.2) but decreases below 6. The K(m) values for both substrates remain constant within the pH range 6-10. At pH values below 6 (for the coenzyme) and above 10 (for both substrate and coenzyme) the Km values increase. In the acidic range this change is attributed to the ionization of two carboxy groups (pK approx. 5.5-6.0) located at the NAD+-binding site of the enzyme active centre. The pH transition in the basic region (pK 10.5 +/- 0.2) has a conformational origin and affects the enzyme's affinity for substrates and anion inhibitors. A similar transition has been observed for formate dehydrogenases from yeast Candida boidinii and Hansenula polymorpha. The results complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme.
Biochem J. 1997 Jan 15;321 ( Pt 2):475-80.   PUBMED

Automated refinement for protein crystallography.
Lamzin VS, Wilson KS
no abstract available
Methods Enzymol. 1997;277:269-305.   PUBMED


Structure of the Val122Ile variant transthyretin - a cardiomyopathic mutant.
Damas AM, Ribeiro S, Lamzin VS, Palha JA, Saraiva MJ
The Val122Ile mutant transthyretin (TTR Ile122) is an amyloidogenic protein which has been described as the major protein component of amyloid fibrils isolated from patients with familial amyloidotic cardiomyopathy (FAC), a disease characterized by cardiac failure and amyloid deposits in the heart. The reasons for the deposition of TTR are still unknown and it is conceivable that a conformational alteration, resulting from the mutation, is fundamental for amyloid formation. The three-dimensional structure of TTR Ile122 was determined and refined to a crystallographic R factor of 15.8% at 1.9 A resolution. The r.m.s. deviation from ideality in bond distances is 0.019 A and in angle-bonded distances is 0.027 A. The presence of two crystallographically independent monomers in the asymmetric unit allowed additional means of estimation of atomic coordinate error. The structure of the mutant is essentially identical to that of the wild-type transthyretin (TTR). The largest deviations occur in surface loops and in the region of the substitution. The protein is a tetramer composed of identical subunits; each monomer has two four-stranded beta-sheets which are extended to eight-stranded beta-sheets when two monomers associate through hydrogen bonds forming a dimer, which is the crystallographic asymmetric unit. The replacement of valine for isoleucine introduces very small alterations in relation to the wild-type protein; nevertheless they seem to confirm a tendency for a less stable tetrameric structure. This would support the idea that the tetrameric structure might be disrupted in amyloid fibrils.
Acta Crystallogr D Biol Crystallogr. 1996 Sep 1;52(Pt 5):966-72. doi: 10.1107/S0907444996003307.   PUBMED

Site-directed mutagenesis of the formate dehydrogenase active centre: role of the His332-Gln313 pair in enzyme catalysis.
Tishkov VI, Matorin AD, Rojkova AM, Fedorchuk VV, Savitsky PA, Dementieva LA, Lamzin VS, Mezentzev AV, Popov VO
Gln313 and His332 residues in the active centre of NAD(+)-dependent formate dehydrogenase (EC, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate-histidine pair in active sites of D-specific 2-hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H-bond to the Gln313 carboxamide.
FEBS Lett. 1996 Jul 15;390(1):104-8.   PUBMED

X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate.
Harutyunyan EH, Kuranova IP, Vainshtein BK, Hohne WE, Lamzin VS, Dauter Z, Teplyakov AV, Wilson KS
The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution. X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric molecule in the asymmetric unit. The upper estimate of the root-mean-square coordinate error is 0.4 A using either the delta A plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion-binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.
Eur J Biochem. 1996 Jul 1;239(1):220-8.   PUBMED

A self-validation technique for protein structure refinement: the extended Hamilton test.
Bacchi A, Lamzin VS, Wilson KS
An extension is proposed for the self-validation Hamilton test [Hamilton (1965). Acta Cryst. 18, 502-510] for crystallographic refinement. The method is based on the statistical F test and evaluates the significance of the R-factor ratio between two refinement protocols. The general case of two refinements carried out with different numbers and types of non-linear restraints is examined. The restraints are considered as extra observations weighted by a coefficient expressing their effective number. There exists a restriction on the weighting coefficients between the two refinements. An empirical method to evaluate the effective number of restraints is provided. The method may allow the detection of unreasonably tight restraints. The expectation value for r.m.s. R(free), given the r.m.s. R, can be estimated. Thus, the significance of the observed drop in R(free) can be assessed. Compared to cross-validation using R(free) [Brunger (1992). Nature (London), 355, 472-474] self-validation has the advantage that it does not require omission of any experimental data. The significance of the improvement obtained by moving from isotropic to anisotropic description of thermal parameters in the refinement of a protein at 1.5 A, resolution is used as an example.
Acta Crystallogr D Biol Crystallogr. 1996 Jul 1;52(Pt 4):641-6. doi: 10.1107/S0907444996001333.   PUBMED

Ribonuclease from Streptomyces aureofaciens at atomic resolution.
Sevcik J, Dauter Z, Lamzin VS, Wilson KS
Crystals of ribonuclease from Streptomyces aureofaciens diffract to atomic resolution at room temperature. Using synchrotron radiation and an imaging-plate scanner, X-ray data have been recorded to 1.20 A resolution from a crystal of native enzyme and to 1.15 A from a crystal of a complex with guanosine-2'-monophosphate. Refinement with anisotropic atomic temperature factors resulted in increased accuracy of the structure. The R factors for the two structures are 10.6 and 10.9%. The estimated r.m.s. error in the coordinates is 0.05 A, less than half that obtained in the previous analysis at 1.7 A resolution. For the well ordered part of the main chain the error falls to below 0.02 A as estimated from inversion of the least-squares matrix. The two independent molecules in the asymmetric unit allowed detailed analysis of peptide planarity and some torsion angles. The high accuracy of the analysis revealed density for a partially occupied anion in the nucleotide binding site of molecule A in the native structure which was not seen at lower resolution. The anisotropic model allowed correction of the identity of the residue at position 72 from cysteine to threonine. Cys72 SG had been modelled in previous analyses with two conformations. The solvent structure was modelled by means of an automated procedure employing a set of objective criteria. The solvent structure for models refined using different programs with isotropic and anisotropic description of thermal motion is compared.
Acta Crystallogr D Biol Crystallogr. 1996 Mar 1;52(Pt 2):327-44. doi: 10.1107/S0907444995007669.   PUBMED

How to escape from model bias with a high-resolution native data set - structure determination of the PcpA-S6 subunit III.
Pignol D, Gaboriaud C, Fontecilla-Camps JC, Lamzin VS, Wilson KS
The structure of procarboxypeptidase A-S6 subunit III, a truncated zymogen E, has been determined by molecular replacement using as search model porcine elastase 1 which, as revealed by crystallographic analysis, contained about 20% of the amino acids in a radically different orientation. Two monoclinic crystal forms were used: the first one diffracts to 2.3 A resolution and contains one molecule per asymmetric unit; the second diffracts to 1.7 A resolution and contains two molecules per asymmetric unit. Molecular replacement and conventional X-PLOR refinement led to a model for which 20% of the chain was ill defined in both crystal forms. To remove the bias introduced by the initial model, an automated refinement procedure [Lamzin & Wilson (1993). Acta Cryst. D49, 129-147] was applied successfully to the second crystal form, which diffracts to high resolution. The resulting dramatic improvement of the electron-density map led to extensive rebuilding of some surface loops. The reliability of the modified model was confirmed by refinement of the first crystal form. For the two forms, the final R factor is 18.8% for data between 8.0 and 2.0 A resolution, and 18.4% for data between 8.0 and 1.7 A, respectively.
Acta Crystallogr D Biol Crystallogr. 1996 Mar 1;52(Pt 2):345-55. doi: 10.1107/S0907444995009528.   PUBMED


Proteins at atomic resolution.
Dauter Z, Lamzin VS, Wilson KS
Experimental advances in data collection, including bright sources, cryogenic cooling and two-dimensional detectors, have made it tractable to record data to beyond 1.2 A for several proteins, yielding high-accuracy models and fine details of structure. For small metalloproteins, atomic-resolution data have enabled ab initio solution of the phase problem.
Curr Opin Struct Biol. 1995 Dec;5(6):784-90.   PUBMED

How nature deals with stereoisomers.
Lamzin VS, Dauter Z, Wilson KS
All natural proteins are composed of L-amino acids and are inherently chiral. The properties of both L- and chemically synthesized D-amino acids are identical except in optically asymmetric interactions. Structural studies of D-I racemic mixtures of crystallographic interest are discussed. The review also gives some recent examples of stereospecificity: how L-proteins deal with L- or D-substrates and how enzymes can function as racemases. Two particular examples of stereoselectivity are then discussed.
Curr Opin Struct Biol. 1995 Dec;5(6):830-6.   PUBMED


Crystallization and preliminary X-ray analysis of a new crystal form of nitrite reductase from Pseudomonas aeruginosa.
Tegoni M, Silvestrini MC, Lamzin VS, Brunori M, Cambillau C
Nitrite reductase from Pseudomonas aeruginosa (EC, a redox enzyme synthesized by the bacterium grown in the presence of nitrate, is a soluble dimer of two identical subunits of 60 kDa, each containing one c and one d1 haem as prosthetic groups. A new crystal from of the Ps. aeruginosa nitrite reductase in the oxidized state, suitable for X-ray structure determination, has been obtained by vapour diffusion at 20 degrees C, in the presence of 10% polyethylene glycol 4000, 50 mM Tris-HCl (pH 8.7), 400 mM NaCl and at a protein concentration of 14 mg/ml. The crystals are dark green elongated tetragonal prisms of dimensions 1.5 mm x 0.2 mm x 0.2 mm for the largest ones. These crystals are tetragonal with space group P4(1(3))2(1)2 and cell dimensions a = b = 128.2 A, c = 172.6 A. They diffract at least up to 2.8 A. Assuming a dimer in the asymmetric unit, the VM value is 2.95 A3/Da (58% of solvent).
J Mol Biol. 1994 Oct 21;243(2):347-50. doi: 10.1006/jmbi.1994.1659.   PUBMED

NAD(+)-dependent formate dehydrogenase.
Popov VO, Lamzin VS
no abstract available
Biochem J. 1994 Aug 1;301 ( Pt 3):625-43.   PUBMED

Dehydrogenation through the looking-glass.
Lamzin VS, Dauter Z, Wilson KS
no abstract available
Nat Struct Biol. 1994 May;1(5):281-2.   PUBMED

High resolution structures of holo and apo formate dehydrogenase.
Lamzin VS, Dauter Z, Popov VO, Harutyunyan EH, Wilson KS
Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 have been refined to R factors of 11.7% and 14.8% at 2.05 and 1.80 A resolution, respectively. The estimated root-mean-square error in atomic co-ordinates is 0.11 A for holo and 0.18 A for apo. X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation. In both crystal forms there is a dimer in the asymmetric unit. Both structures show essentially 2-fold molecular symmetry. NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears. This completes formation of the enzyme active centre in holo FDH. NAD is bound in the cleft separating the domains and mainly interacts with residues from the co-enzyme binding domain. In apo FDH these residues are held in essentially the same conformation by water molecules occupying the NAD binding region. An azide molecule is located near the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding site. There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active centre after NAD has already bound. The structure of the active site and a hypothetical catalytic mechanism are discussed. Sequence homology of FDH with other NAD-dependent formate dehydrogenases and some D-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure.
J Mol Biol. 1994 Feb 25;236(3):759-85. doi: 10.1006/jmbi.1994.1188.   PUBMED


Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 A resolution.
Betzel C, Dauter Z, Genov N, Lamzin V, Navaza J, Schnebli HP, Visanji M, Wilson KS
The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three-dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the 'scissile' bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%.
FEBS Lett. 1993 Feb 15;317(3):185-8.   PUBMED

Automated refinement of protein models.
Lamzin VS, Wilson KS
An automated refinement procedure (ARP) for protein models is proposed, and its convergence properties discussed. It is comparable to the iterative least-squares minimization/difference Fourier synthesis approach for small molecules. ARP has been successfully applied to three proteins, and for two of them resulted in models very similar to those obtained by conventional least-squares refinement and rebuilding with FRODO. In real time ARP is about ten times faster than conventional refinement. In its present form ARP requires high (2.0 A or better) resolution data, which should be of high quality and a starting protein model having about 75% of the atoms in roughly the correct position. For the third protein at 2.4 A resolution, ARP was significantly less powerful but nevertheless gave definite improvement, in the density map at least.
Acta Crystallogr D Biol Crystallogr. 1993 Jan 1;49(Pt 1):129-47. doi: 10.1107/S0907444992008886.   PUBMED


Crystal structure of NAD-dependent formate dehydrogenase.
Lamzin VS, Aleshin AE, Strokopytov BV, Yukhnevich MG, Popov VO, Harutyunyan EH, Wilson KS
The ternary complex of NAD-dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 (enzyme-NAD-azide) has been crystallised in the space group P2(1)2(1)2(1) with cell dimensions a = 11.60 nm, b = 11.33 nm, c = 6.34 nm. There is 1 dimeric molecule/asymmetric unit. An electron density map was calculated using phases from multiple isomorphous replacement at 0.30 nm resolution. Four heavy atom derivatives were used. The map was improved by solvent flattening and molecular averaging. The atomic model, including 2 x 393 amino acid residues, was refined by the CORELS and PROLSQ packages using data between 1.0 nm and 0.30 nm excluding structure factors less than 1 sigma. The current R factor is 27.1% and the root mean square deviation from ideal bond lengths is 4.2 pm. The FDH subunit is folded into a globular two-domain (coenzyme and catalytic) structure and the active centre and NAD binding site are situated at the domain interface. The beta sheet in the FDH coenzyme binding domain contains an additional beta strand compared to other dehydrogenases. The difference in quaternary structure between FDH and the other dehydrogenases means that FDH constitutes a new subfamily of NAD-dependent dehydrogenases: namely the P-oriented dimer. The FDH nucleotide binding region of the structure is aligned with the three dimensional structures of four other dehydrogenases and the conserved residues are discussed. The amino acid residues which contribute to the active centre and which make contact with NAD have been identified.
Eur J Biochem. 1992 Jun 1;206(2):441-52.   PUBMED


NAD-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. I. Amino acid sequence].
Popov VO, Shumilin IA, Ustinnikova TB, Lamzin VS, Egorov TsA
The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 is determined. The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD. To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion. The peptides obtained were purified to homogeneity and characterized.
Bioorg Khim. 1990 Mar;16(3):324-35.   PUBMED

NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. II. Enzyme conformation at 3.0 A resolution].
Lamzin VS, Aleshin AE, Popov BO, Arutiunian EG
Three heavy atom isomorphous derivatives were used for the X-ray analysis of the holo form of NAD-dependent bacterial formate dehydrogenase (ternary complex enzyme-NAD-azide) at 3.0 A resolution. The enzyme subunit contains a catalytic and a coenzyme binding domain, with the active centre and the coenzyme binding site in the cleft between the domains. The polypeptide chain's fold and the position of 393 C alpha-atoms were determined. The secondary structure of the formate dehydrogenase was resolved. The structure of the NAD-binding domain is shown to be similar to that of other NAD-dependent enzymes.
Bioorg Khim. 1990 Mar;16(3):336-44.   PUBMED

NAD-dependent formate dehydrogenase of methylotrophic bacteria Pseudomonas sp. 101. III. Comparative analysis].
Lamzin VS, Aleshin AE, Shumilin IA, Ustinnikova TB, Egorov TsA, Arutiunian EG, Popov VO
The comparative analysis of the primary and tertiary structures of NAD-dependent bacterial formate dehydrogenase (FDH) from methylotrophic bacterium Pseudomonas sp. 101 and a number of structurally characterized NAD-dependent dehydrogenases were performed. FDH has a highly conservative fold of the coenzyme binding domain. Position of the symmetry axis in the FDH molecule relative to the beta-sheets of its coenzyme binding domain with the respective sequences of the other NAD-dependent enzymes was performed on the basis of the spatial homology between these structures. Only one of the three amino acid residues previously thought to be conserved in the coenzyme binding domains of NAD-dependent dehydrogenases is preserved in the FDH molecule (Asp-221). Two glycine residues found in all previously studied dehydrogenases are substituted in FDH by Ala-198 and Pro-256, respectively. Position of the essential thiol of FDH (Cys-255) in the protein structure was established. It is suggested that Cys-255 is situated on or near polypeptide locus taking part in the conformational changes of the protein in the course of the catalysis.
Bioorg Khim. 1990 Mar;16(3):345-57.   PUBMED

Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase.
Bogdanova AV, Cherednikova TV, Egorov TA, Harutyunyan EG, Kurochkina NA, Lamzin VS, Savitskiy AP, Shumilin IA, Popov VO
A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 A resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.
FEBS Lett. 1990 Jan 29;260(2):297-300.   PUBMED


X-ray analysis of bacterial NAD-dependent formate dehydrogenase at resolution of 5 angstroms].
Aleshin AE, Lamzin VS, Devedzhiev IaD, Rubinskii SV, Popov VO
no abstract available
Dokl Akad Nauk SSSR. 1987;294(4):973-6.   PUBMED


Conformation changes in bacterial formate dehydrogenase during complex-formation with cofactor and its analogs].
Lamzin VS, Asadchikov VE, Popov VO, Egorov AM, Berezin IV
no abstract available
Dokl Akad Nauk SSSR. 1986;291(4):1011-4.   PUBMED

Crystallization and preliminary X-ray diffraction study of bacterial formate dehydrogenase].
Devedzhiev Ia, Moroz OV, Arutiunian EG, Lamzin VS, Egorov AM
no abstract available
Dokl Akad Nauk SSSR. 1986;286(3):757-60.   PUBMED


Modification of bacterial formate dehydrogenase by a mercarbide electron-dense label. X-ray determination of the space between active centers of the enzyme].
Lamzin VS, Asadchikov VE, Egorov AM, Marakushev SA, Popov VO
no abstract available
Dokl Akad Nauk SSSR. 1985;281(3):712-5.   PUBMED


no abstract available

no abstract available

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