Předmět Structural biology methods practical (C7271)

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Materiál	Typ	Datum	Počet stažení

Cíl

This practical will allow students to gain hands on experience with macromolecular structure determination. Some of the excercises will reinforce understanding of basic theoretical concepts while others will involve use of expert computer programs that allow calculation of macromolecular structures. Structure of the practical mirrors that of the ”Structural Biology Methods” lecture series: X-ray crystallography (9 practicals), cryo-electron microscopy (cryo-EM) (3 practicals), and atomic force microscopy (AFM) (1 practical).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

Week 1: Introduction, crystals and symmetry I., and X-rayso Introduction to the courseo Excursion of protein crystallization and X-ray diffraction equipment at CEITECo Practical Protein Crystallization - lysosymeo 2D symmetry exercise (finish as homework)• Read chapters 1, 2, 3 and 16 from the textbook.Week 2: Crystals and Symmetry II. (continued) and the Theory of X-Ray Diffractiono Excercises on Crystal Systems and Matthew’s coefficiento Excercises on goniometric and exponential functions; wave addition; Argand diagram• Read chapters 3 and 4 from the textbook.Week 3: The Theory of X-Ray Diffraction by a Crystal I.o Derivation of Bragg’s law, calculation of unit cell parameters from diffraction patterno Drawing Reciprocal Lattice and Ewald Construction• Read chapters 4 from the textbook.Week 4: The Theory of X-Ray Diffraction by a Crystal II.o Exercise on comparison of F(hkl) and F(h ̄k ̄l ̄); Symmetry in the Diffraction Pattern; Integral Reflection Conditions for Centered Lattices• Read chapter 4 from the textbook.Week 5: Average Reflection Intensity, Distribution of Structure Factor Data, Special Forms of the Structure Factor.o Computer exercise – data indexing, integration and scaling• Read chapters 5 and 6 from the textbook.Week 6: The Solution of the Phase Problem by the Isomorphous Replacement Methodo Computer exercise – calculation of Patterson function, identification of heavy atoms peaks in a map, calculation of protein density• Read chapter 7 from the textbook.Week 7: Phase Improvemento Computer exercise – real space averaging, solvent flattening• 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 Computer exercise – calculation of macromolecular density based on phases from MADo Computer exercise – rotation functiono Computer exercise – translation 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 Computer exercise – fully automated molecular replacemento Computer exercise – model building in Cooto Computer exercise – R-Factors; The Ramachandran Plot; Stereochemistry Check• Read chapters 10, 12, 13, 14 and 15 from the textbook.Week 10: Electron Microscopy of Macromolecular Assemblieso Visit to cryo-EM facility at CEITEC, observe sample preparation, sample insertion into microscope, and microscope operation• Read chapter 2 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”Week 11: Multivariate Data Analysis and Classification of Imageso Computer exercise – particle boxing and CTF correction• Read chapters 3 and 4 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”Week 12: Three-Dimensional Reconstructiono Computer exercise – calculation of 3D reconstruction• Read chapter 5 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”Week 13: Atomic Force Microscopyo Immobilization of biomolecules on the surfaceo Measurement of the biomolecules with AFM microscope• Read review Trache A, Meininger GA. Atomic force microscopy (AFM); Curr Protoc Microbiol. 2008; Chapter 2.

Literatura

povinná literaturaJoachim Frank. (2010) “Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State”, 3rd edition; ISBN-13: 9780195182187; Oxford Scholarship OnlineJan 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, USATrache A, Meininger GA. Atomic force microscopy (AFM); Curr Protoc Microbiol. 2008; Chapter 2.

Požadavky

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

Garant

prof. RNDr. Zdeněk Glatz, CSc.

Vyučující

Liya MukhamedovaMgr. Pavel Plevka, Ph.D.Mgr. Marta ŠiborováMgr. Karel ŠkubníkHoa Khanh Tran Kiem, M.Sc.