043CM - BIOCRYSTALLOGRAPHY AND ELECTRON MICROSCOPY 2024
Section outline
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Introduction to structural biology: the importance of structure-property relationships. Protein databases. Software for visualizing protein structures.
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Hypothetical mechanism of the enzyme, based on the crystallographic structures of the protein obtained in different states.
The video was prepared with UCSF Chimera starting from coordinate files available on PDB.
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This video was generated following the procedure described in the last slides of the introduction.
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Basics of protein structure: primary, secondary, tertiary, and quaternary structure, with examples. Geometric constraints and degrees of freedom. Post-translational modifications and co-factors. Motifs and domains. Membrane proteins.
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Expression of recombinant proteins for structural studies. Design of the protein construct. Selection of a suitable expression system. Main molecular biology techniques used in structural biology.
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Bioinformatics resource operated by the SIB Swiss Institute of Bioinformatics. It includes various bioinformatic tools, among which databases and online software for structural biology, medicinal chemistry, genomics, proteomics, and systems biology.
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This protein sequence database includes annotations about localization, function, structure, post-translational modifications, etc. The website also contains links to bioinformatics tools for sequence alignment (Align) and primary sequence homology searches (Blast).
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Purification techniques. Methods to increase the stability, conformational homogeneity, and crystallizability of a protein. Evaluation of the quality and purity of the sample. Analysis of the conformational stability. Analysis of the oligomeric state of the protein.
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Crystallization of soluble and membrane proteins. Thermodynamic and kinetic perspectives on protein crystallization. Main crystallization techniques and optimization strategies, including co-crystallization and soaking.
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Protein crystals. Symmetry point elements. Crystal symmetry. Crystal families and systems. Bravais lattices. Screw axes: rotation and translation. Fractional coordinates. Space groups. International Crystallographic Tables. Non-crystallographic symmetry. Miller indices and reciprocal lattice.
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X-ray diffraction: basic concepts about the physics of diffraction. Bragg's law. Ewald sphere and reciprocal lattice. Symmetry of the reciprocal lattice. Laue classes. Systematic absences. Anomalous scattering.
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Instruments: X-ray source, optical components of the diffractometer, goniometer, detector. Crystal cooling for cryocrystallography. Crystal centering. The diffraction experiment. Optimization of the data collection parameters. Indexing. Integration. Space group determination. Scaling and merging. Data quality evaluation. Radiation damage. Twinning. (Notes on Serial Femtosecond Crystallography, SFX, with X-ray Free Electron Laser, X-FEL.)
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Structure factors and electron density. The Fourier transform and its properties. Convolution. The Patterson function. Moduli and phases. The phase problem. Matthews volume. Methods for solving the phase problem for protein crystals.
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Phasing the diffraction data through Molecular Replacement. Bulk-solvent correction. Density Modification techniques. Solvent flattening/flipping. Histogram matching. Non-crystallographic symmetry: identification of rotation and translation operators. Density averaging.
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Phasing techniques based on isomorphous replacement or anomalous scattering. Data preparation. Determination of the position of the heavy atom or the anomalous scatterer: direct methods and Patterson methods. SIR: Single Isomorphous Replacement. SAD: Single-wavelength Anomalous Diffraction. Resolution of the ambiguity on the phases determined by SIR or SAD. MIR: Multiple Isomorphous Replacement. MAD: Multiple-wavelength Anomalous Diffraction. Electron density maps. Resolution and structural elements.
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From phases to electron density. Model building. Refinement in reciprocal space. Observations and parameters. Restraint and constraint. Minimization functions for parameter optimization. Refinement protocol. Side chain conformations. Solvent and ligand modeling. Rfree and Rwork.
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Importance of structure validation. Bayesian statistics applied to crystallographic model validation. Global and local parameters. Geometry analysis: bond angles and distances, Ramachandran diagram, repulsive interactions (clashes). Analysis of electron density and correlation parameters for single residues. Validation of the ligand occupancy and geometry.
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Read the paper and spot significant crystallographic mistakes!
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... File PDF
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Differences between electrons and X-rays. The main electron microscopy techniques used in structural biology. The electron microscope: source, electromagnetic lenses, compustage, detector. Direct detection cameras and their characteristics. Physical principles of image formation in the microscope: "weak phase object" approximation, effect of the objective lens (astigmatism and spherical aberration), wave function in the back focal plane, image formation. Contrast Transfer Function (CTF) and its correction with phase flipping and Wiener filter. Phase plates.
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Sample preparation: negative staining and cryo-EM. Sugar embedding. Sample preparation for single-particle cryo-EM. Electron microscopy techniques for high-resolution structural biology. Data collection: diffraction images and patterns. Electron crystallography. Preparation of two-dimensional crystals. Diffraction data collection: the reciprocal space of two-dimensional crystals. Analysis of diffraction data. Analysis of electron microscopy images of two-dimensional crystals: filtering, unbending, CTF correction and merging to obtain the projection map. Two examples of structures obtained with electron crystallography: bacteriorhodopsin and aquaporin-0.
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Signal and noise in electron microscopy images. Sampling theorem. Fundamental parameters of the EM imaging. Low-pass filtering. Particle selection and masking. Noise reduction by averaging. Automatic particle selection. Alignment: rotation and translation. Sample heterogeneity. Classification: Principal Component Analysis and two-dimensional image classification methods. Ascending hierarchical classification method. K-means method. Examples of classification in negative staining and cryo EM.
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Reconstruction of the three-dimensional model from two-dimensional images. Euler angles and projections. Projection theorem. (1) Determination of the orientation of the particles: tomography, Random Conical Tilt method, common line method. (2) Reconstruction. CTF correction. Heterogeneity. Method based on Bayesian statistical analysis (Relion software).
Validation of the structural model. Two examples of incorrect reconstruction. Resolution. Some examples of proteins whose structure has been determined at high resolution with cryo-EM technique.
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Small home-made guide for beginners.
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