Highlights in atomic and ultra-high resolution protein crystallography
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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 chemical environment around the active-site residues. Together with the occurrence of multiple-residue conformers, they reflect spatial rearrangement throughout the deacylation pathway.
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Substrate attachment and detachment in trypsin. The binding state is reflected in the directional motion.
Atoms are represented as 1 Å spheres, the colour is derived from the principal direction of motion. The vector components were translated into RGB colour code: RED=X, GREEN=Y, BLUE=Z. Below: the corresponding distance changes. |
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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.
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Advantages of high resolution phasing: MAD to atomic resolution
Schmidt A., Gonzalez A., Morris R.J., Costabel M., Alzari P.M., Lamzin V.S. (2002)
Advantages of high-resolution phasing: MAD to atomic resolution. Acta Cryst. D, 58, 1433-41.
(medline)
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 Å. 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 Å 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.
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| D/B plot (electron density at atomic centre vs. B factor) for the atoms in the CelA MAD struture. Atom types can clearly be distinguished. |
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| Averaged electron density over peptides showing the different bond orders and -in the difference map- hydrogen atoms. |
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Atomic (0.94 Å) Resolution Structure of an inverting Glycosidase in Complex with Substrate
Guerin, D.M.A., Lascombe, M.B., Costabel, M., Souchon, M.H., Lamzin, V. S., Beguin, P. and Alzari, P. (2002).
Atomic (0.94 Å) Resolution Structure of an inverting Glycosidase in Complex with Substrate,
J.Mol.Biol 316, 1061-1069.
(medline)
The crystal structure of Clostridium thermocellum endoglucanase CelA in complex with cellopentaose has been determined at 0.94 Å 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.
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| Electron density in the active site of CelA. The substrate is yellow, the product violet. Map: 3Fo-2Fc |
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| ORTEP plot for the substrate in CelA, showing anisotropy and occupancies in the subsites |
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Atomic resolution data reveal flexibility in the structure of RNAse Sa
Sevcik J., Lamzin V.S., Dauter Z., Wilson K.S. (2002) Atomic resolution data reveal flexibility in the structure of RNase Sa
Acta Cryst. D, 58, 1307-13.
(medline)
Ribonuclease from Streptomyces aureofaciens, the bacterial source for the industrial production of chlorotetracycline, is a guanylate endoribonuclease (RNase Sa; EC 3.1.27.3) 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 Å) using room-temperature data. Here, the RNase Sa structure refined against 1.0 Å 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 Å, with a maximum of 2.5 Å. 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.
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| Schematic representation of a disordered part in the RNase Sa structure. |
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| Electron density for the disordered region at cryo (left) and at room temperature. |
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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
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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.
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| Electron density for histidine 12 in RNAse A. Red maps indicate acidic, blue maps basic pH. Difference maps Fo(1/2)Eo(1/2) - Fc(1/2)Ec(1/2) are contoured at 1.6 sigma. |
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X-ray data have been recorded to 1.0 Å 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 Å, 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.
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Protonation state of a histidine side-chain in Fusarium solanii cutinase |
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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.
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| Thermal ellipsoids for the catalytic triad and the substrate (left) or inhibitor (right), indicating directional movement fro the covalent inhibitor complex. |
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| His 56 of the catalytic triad with deformation electron density (left) and charge density (right) map. His 56 is non-protonated. |
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Atomic (1 Å) 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.
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| Electron density in the active site of LADH |
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| Crystal (yellow) and theoretical ab initio structures (cyan) of the nicotinamide ring of NADH under the ingluence of a water molecule (a) and a hydroxyl ion (b) |
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Observation of bonding electrons
Lamzin, V. S., Morris, R. J., Dauter, Z., Wilson, K. S., and Teeter, M. M. (1999).
Experimental observation of bonding electrons in proteins, J.Biol.Chem
274, 20753-20755.
(medline)
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.
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| Averaged 3Fo-2Fc map |
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| Averaged difference map |
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Valence electron distribution in Crambin
Jelsch, C., Teeter, M. M., Lamzin, V. S., Pichon-Pesme, V., Blessing, R. H., and
Lecomte, C. (2000). Accurate protein crystallography at ultra-high resolution:
valence electron distribution in crambin, Proc.Nat.Acad.Sci 97, 3171-3176.
(medline)
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 Å) 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.
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| Theoretical(A) and observed (B) electron density in Crambin |
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Last update: October 15th, 2005