Předmět Structural biology methods (C7270)

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Materiál	Typ	Datum	Počet stažení

Cíl

This course aims to provide understanding of theoretical background and practical aspects of X-ray crystallography (9 lectures), cryo-electron microscopy (cryo-EM) (3 lectures), and atomic force microscopy (AFM) (1 lecture). Students will learn methodological approaches allowing determination of macromolecular structures with atomic details. Both X-ray crystallography and cryo-EM use the concepts of reciprocal space and Fourier transform to allow calculation of electron density maps that show shapes of macromolecules. Cryo-EM part of the course is limited to three lectures, however, the necessary mathematical concepts will be introduced in preceding X-ray crystallography lectures. AFM complements the course since it allows studies of physical properties and of composition of macromolecular complexes with nanometer resolution.By the end of this course, students should be able to do the following:• Understanding of principles allowing the use of X-ray crystallography, cryo-EM, and AFM.• Undestanding of theory behind:o Properties of X-ray radiation that make it suitable to study (macro)molecular structureso Diffraction of lighto Crystallographic space group symmetrieso Approaches to resolve phase problem in crystallographyo Use of electrons to display objects with high magnification and fine detailo Calculation of three-dimensional reconstruction from two-dimensional projections of an objecto Analysis of macromolecular surfaces by AFM• Use of structural biology approaches to study molecular processes necessary for the existence of life.• Students will be able to describe how structural methods allowed fundamental discoveries in biology such as:o Synthesis of proteins by ribosomeo Functions of Rubisco a photosystems 1 and 2o Structures of viruses and development of antiviral therapeutics• Students will gain basic practical experience in use of the methods.Knowledge gained in the course can be used in both basic and applied research to study structures of macromolecules and their complexes. Students will learn skills that will allow them to apply for jobs with local high-tech companies such as Tescan and FEI.

Osnova

Course OutlineWeek 1: Introduction, crystals and symmetry I., and X-rayso Introduction to the courseo Crystallizing a Proteino Practical Protein Crystallization (Chapter 16)o X-Ray Sources and Detectors; Crystals and symmetry I.• Read chapters 1, 2, 3 and 16 from the textbook.Week 2: Crystals and Symmetry II. (continued) and the Theory of X-Ray Diffractiono Asymmetric Unit; Point Groups; Crystal Systems; Characterization of the Crystals; Matthew’s coefficiento Waves and Their Addition; Argand diagram; Exponential Terms• Read chapters 3 and 4 from the textbook.Week 3: The Theory of X-Ray Diffraction by a Crystal I.o A System of Two Electrons; Scattering by an Atom; Scattering by a Unit Cell; Scattering by a Crystal; Diffraction Conditions; Reciprocal Lattice and Ewald Construction• Read chapters 4 from the textbook.Week 4: The Theory of X-Ray Diffraction by a Crystal II.o The Temperature Factor; Calculation of the Electron Density ρ(x y z); Comparison of F(hkl) and F(h ̄k ̄l ̄); Symmetry in the Diffraction Pattern; Integral Reflection Conditions for Centered Lattices; Intensity Diffracted by a Crystal; The Polarization Factor; Scattering by a Plane of Atoms; Choice of Wavelength, Size of Unit Cell, and Correction of the Diffracted Intensity• Read chapter 4 from the textbook.Week 5: Average Reflection Intensity, Distribution of Structure Factor Data, Special Forms of the Structure Factor.o Average Intensity; Wilson Plots; The Distribution of Structure Factors F and Structure Factor Amplitudes |F|; Crystal Twinningo The Unitary Structure Factor; The Normalized Structure Factor; o Phase problem recapitulation• Read chapters 5 and 6 from the textbook.Week 6: The Solution of the Phase Problem by the Isomorphous Replacement Methodo The Patterson Function; Convolution; The Isomorphous Replacement Method; Effect of Heavy Atoms on X-ray Intensities; Determination of the Heavy Atom Parameters from Centrosymmetric Projections; Parameters of Heavy Atoms Derived from Acentric Reflections; The Difference Fourier Summation o Anomalous Scattering; The Anomalous Patterson Summation; Refinement of the Heavy Atom Parameters Using Preliminary Protein Phase Angles; Protein Phase Angles; The Remaining Error in the Best Fourier Map; The Single Isomorphous Replacement Method;• Read chapter 7 from the textbook.Week 7: Phase Improvemento The OMIT Map With and Without Sim Weighting; Solvent Flattening; Non-crystallographic Symmetry and Molecular Averaging; Histogram Matching; wARP: Weighted Averaging of Multiple-Refined Dummy Atomic Models; Further Considerations Concerning Density Modification• Read chapter 8 from the textbook.Week 8: Anomalous Scattering in the Determination of the Protein Phase Angles and the Absolute Configuration and Molecular Replacement I.o Protein Phase Angle Determination with Anomalous Scattering; Improvement of Protein Phase Angles with Anomalous Scattering; The Determination of the Absolute Configuration; Multiple- and Single-Wavelength Anomalous Diffraction (MAD and SAD);o The Rotation Function;• Read chapters 9 and 10 from the textbook.Week 9: Molecular Replacement II., Laue Diffraction, Refinement of the Model Structure, The Combination of Phase Information, Checking for Gross Errors and Estimating the Accuracy of the Structural Model.o The Translation Function; AMORE; Rigid-Body Refinement;o The Accessible Region of Reciprocal Space; The Multiple Problem; Unscrambling of Multiple Intensities; The Spatial Overlap Problem; Wavelength Normalization;o The Mathematics of Refinement; The Principle of the Fast Fourier Transform Method; Specific Refinement Methods;o Phase Information from Isomorphous Replacement; Phase Information from Anomalous Scattering; Phase Information from Partial Structure Data, Solvent Flattening, and Molecular Averaging; Phase Information from SADo R-Factors; The Ramachandran Plot; Stereochemistry Check; The 3D–1D Profile Method; Quantitative Estimation of the Coordinate Error in the Final Model;• Read chapters 10, 12, 13, 14 and 15 from the textbook.Week 10: Electron Microscopy of Macromolecular Assemblieso Principle of the Transmission Electron Microscope; The Weak-Phase Object Approximation; The Contrast Transfer Theory; Determination of the Contrast Transfer Function; Computational Correction of the Contrast Transfer Function;• Read chapter 2 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”Week 11: Multivariate Data Analysis and Classification of Imageso Alignment Methods; Averaging and Global Variance Analysis; Resolution; Validation of the Average Image;o Theory of Correspondence Analysis; Classification• Read chapters 3 and 4 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”Week 12: Three-Dimensional Reconstructiono General Mathematical Principles; The Rationales of Data Collection: Reconstruction Schemes; Overview of Existing Reconstruction Techniques; Reference-Based Methods and Refinement; Contrast Transfer Function and Fourier Amplitude Correction; Resolution Assessment• Read chapter 5 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”Week 13: Atomic Force Microscopyo General principles of Scanning Probe Microscopy; Basic scheme and principles of Atomic Force Microscope; Study of biological objects by Atomic Force Microscopy; Immobilization of biomolecules and other biological objects; AFM spectroscopy – Force-Distance curves; Evaluation of AFM data – images; curves and combined data.• Read review Trache A, Meininger GA. Atomic force microscopy (AFM); Curr Protoc Microbiol. 2008; Chapter 2.

Literatura

povinná literaturaTrache A, Meininger GA. Atomic force microscopy (AFM); Curr Protoc Microbiol. 2008; Chapter 2.Jan Drenth. (2007) “Principles of Protein X-Ray Crystallography”, 3rd edition; ISBN: 978-0-387-33334-2; JSpringer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USAo Joachim Frank. (2010) “Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State”, 3rd edition; ISBN-13: 9780195182187; Oxford Scholarship Online

Požadavky

This lecture series is intended for students of biology (general biology, molecular biology and genetics, specialised biology, biochemistry) and for other students interested in macromolecular structure determination. This lecture series is complemented by practical series “Structural Biology Methods Practical“. The two courses are supposed to be read during the same semester.

Garant

prof. RNDr. Zdeněk Glatz, CSc.

Vyučující

Mgr. Pavel Plevka, Ph.D.