Seminar Colour Guide:
External Faculty | External Postdoc | Company Representative Science and Society EMBL Distinguished Visitor Lecture Vision2020 Lecture Series Molecular Medicine Seminar | EIPOD Seminar | PSB Seminar | TAC Seminar Hamburg Speaker EMBL-La Sapienza Lecture
Abstract: The macromolecular crystallography P13 beamline is part of the European Molecular Biology Laboratory Integrated Facility for Structural Biology at PETRA III (DESY, Hamburg, Germany) and in user operation since mid 2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallographic data acquisition exploiting a wide range of elemental absorption edges for experimental phase determination. An adaptive Kirk-Patrick-Baez focussing system provides an X-ray beam with a high photon flux (up to 6*1012 ph/s at 9 keV), a low beam divergence (0.2 mrad (H) × 0.15 mrad (V)) and rapidly (few minutes) tunable focus size (30 μm to 150 μm, horizontally, 24 μm to 70 μm, vertically) to adapt to diverse experimental situations. The ARINAX MD2 diffractometer and the small focus size of the beam facilitate collection of accurate diffraction data from small crystals (down to 5 µm linear dimensions) and allow precise helical scans on needle-shaped crystals. The mini-κ goniometer attached to the MD2 spindle axis allows crystal re-orientation for optimized anomalous data collection.
Data collections at energies as low as 4 keV (3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux in particular at low energy (up to 1011 ph/sec at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4*1011 ph/s at 17.5 keV) combined with a large area detector mounted on a 2θ-arm allows data collection to sub-atomic resolution (0.55 Å). Automated sample mounting is available by means of the robotic sample changer 'MARVIN' with a duty-cycle of less than one minute and a dewar capacity of 160 samples. In close proximity to the beamline, laboratories have been set up for sample preparation and characterization; a laboratory specifically equipped for on-site heavy atom derivatization with a library of more than 150 compounds is available to beamline users.
The capabilities of the beamline are demonstrated by examples of sulphur-SAD phasing enabled by X-ray beams adjusted optimally both in terms of beam size and X-ray energy and by successful structure solutions from small crystals.
|External Faculty Speaker||Abstract: We work at the interface of systems biology and molecular mechanism. On one hand we develop and utilize high-throughput quantitative approaches that reveal functional interactions between genes at a whole cell level. On the other hand, we zoom into these networks to understand how different functional modules are interconnected, often at a detailed mechanistic level. Here I will present how we use such approaches to shed light into gene function and pathway organization, to understand the action of drugs and their interplay when combined, and to probe how protein machineries operate at the bacterial cell envelope- how they are organized, how they coordinate their actions and how the cell senses when they are malfunctioning. We have also recently moved our approaches to the host-pathogen interface and the dynamic microbial communities formed in our gut. Our main goals are to: a) elucidate pathways Salmonella uses to hijack its host machinery and b) to probe how gut microbial communities are formed, how they react to nutrition and pharmaceuticals, and how their composition and characteristics affects our health.|
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Abstract: The study of the three-dimensional structure of biological macromolecules and assemblies, and their organization in the cellular context is of utmost importance for understanding many biological processes. For example the ribosome synthesizes protein from nucleic acid and its function is intimately related to its structure and changes therein. Multiple ribosomes have been observed in a cellular context processing the same strand of mRNA in order to synthesize protein efficiently.
Cryo-electron microscopy (cryoEM) and electron tomography (ET) have become indispensable tools for molecular and cellular structural biology. In recent years technological advances such as the director electron detector have led to a breakthrough in the level of observable detail. We are now witnessing a period of dramatic expansion in the number of people using cryoEM techniques for structure determination. Moreover the capabilities of the technique are being pushed to the very limits, for example, by using phase plate technology to examine low molecular weight samples and by exploiting image-processing techniques to obtain 3D snapshots of structurally variable samples.
The open and public access to structural data is of utmost importance for validation, development, testing and training. The Electron Microscopy Data Bank (EMDB) archive was established at the European Bioinformatics Institute (EBI) in 2002 and is the authoritative source for 3DEM data. Against the backdrop of technological advances, EMDB has experienced rapid growth and now contains over 3600 structures. In 2014 the Protein Data Bank in Europe (PDBe) started EMPIAR the electron microscopy pilot image archive to store raw image data related to EMDB structures. The challenge here has been in dealing with the storage and transfer of large datasets. EMPIAR now holds circa 50 datasets and approximately 25 TB/month is downloaded on average. In this talk I will present the status of the archives and of on-going initiatives related to the archives such as the EMDataBank Validation Challenges, efforts underway on the topics of validation, data-mining and integration, and future challenges and opportunities.
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|External Faculty Speaker||Abstract: Influenza A virus is an enveloped negative strand RNA virus. Its outer envelope consists of the lipid membrane with incorporated glycoproteins and proton channel M2. The inner envelope of the virion is a membrane-associated scaffold of matrix protein M1, which contacts both the viral RNP and the lipid envelope. Formation and disintegration of the protein scaffold are essential processes for influenza replication and infection. Both involve interaction of M1 with the lipid membrane; both are controlled by pH. We investigate the physico-chemical mechanism of these processes using the combination of electrochemical and fluorescent measurements with AFM. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. Acidification drives conformational changes in M1 molecules and increase of their charge leading to partial desorption due to increased repulsion between M1 monomers still stuck to the membrane. This repulsive force could generate tension for membrane rupture, as it was demonstrated for lipid vesicles coated with M1. Thus, electrostatic forces could explain M1 protein scaffold disintegration at low pH and most likely stretch the lipid membrane, promoting fusion pore widening for RNP release. Performing the measurements of M1 adsorption at different ionic strengths of the solution, we estimated the charge of M1 in the concerned range of pH. Our results show that at pH of late endosome scaffold protein M1 significantly changes its charge meaning that electrostatics could be the main driving force in disassembly of Influenza A virus protein envelope. On the other hand, we demonstrated that assembly of M1 molecules in helices should occur in a pH-independent manner. Modelling these processes using Derjagin-Landau-Verwey-Overbeek theory (DLVO) allows us to estimate the energy of M1-M1 interactions.|
|Seminar given by an external postdoc|
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