C7271 Structural biology methods practical

Faculty of Science
autumn 2017
Extent and Intensity
0/2. 2 credit(s) (plus extra credits for completion). Type of Completion: z (credit).
Teacher(s)
Liya Mukhamedova (seminar tutor)
Mgr. Pavel Plevka, Ph.D. (seminar tutor)
Mgr. Marta Šiborová (seminar tutor)
Mgr. Karel Škubník (seminar tutor)
Hoa Khanh Tran Kiem, M.Sc. (seminar tutor)
Supervisor
prof. RNDr. Zdeněk Glatz, CSc.
Department of Biochemistry - Chemistry Section - Faculty of Science
Supplier department: Pavel Plevka Research Group - Centre for Structural Biology - Central European Institute of Technology
Timetable
Mon 18. 9. to Fri 15. 12. Mon 13:30–15:00 A35-211
Prerequisites
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.
Course Enrolment Limitations
The course is also offered to the students of the fields other than those the course is directly associated with.
Fields of study the course is directly associated with
Course objectives
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 structures
o Diffraction of light
o Crystallographic space group symmetries
o Approaches to resolve phase problem in crystallography
o Use of electrons to display objects with high magnification and fine detail
o Calculation of three-dimensional reconstruction from two-dimensional projections of an object
o 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 ribosome
o Functions of Rubisco a photosystems 1 and 2
o 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.
Syllabus
  • Week 1: Introduction, crystals and symmetry I., and X-rays
  • o Introduction to the course
  • o Excursion of protein crystallization and X-ray diffraction equipment at CEITEC
  • o Practical Protein Crystallization - lysosyme
  • o 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 Diffraction
  • o Excercises on Crystal Systems and Matthew’s coefficient
  • o 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 pattern
  • o 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 Method
  • o 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 Improvement
  • o 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 MAD
  • o Computer exercise – rotation function
  • o 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 replacement
  • o Computer exercise – model building in Coot
  • o 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 Assemblies
  • o 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 Images
  • o Computer exercise – particle boxing and CTF correction
  • • Read chapters 3 and 4 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”
  • Week 12: Three-Dimensional Reconstruction
  • o Computer exercise – calculation of 3D reconstruction
  • • Read chapter 5 from “Three-Dimensional Electron Microscopy of Macromolecular Assemblies”
  • Week 13: Atomic Force Microscopy
  • o Immobilization of biomolecules on the surface
  • o Measurement of the biomolecules with AFM microscope
  • • Read review Trache A, Meininger GA. Atomic force microscopy (AFM); Curr Protoc Microbiol. 2008; Chapter 2.
Literature
    required literature
  • 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
  • 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, USA
  • Trache A, Meininger GA. Atomic force microscopy (AFM); Curr Protoc Microbiol. 2008; Chapter 2.
Teaching methods
Course Format
In this practical, you will be acquiring knowledge in two main ways: (1) solving of problems that will reinforce your understanding of theoretical approaches required for macromolecular structure determination and (2) use of expert softvare for calculation of macromolecular structures.

Learning suggestions
o Read the readings before the day for which they are assigned. This will help you to acquire knowledge gradually in small chunks.
o When reading summarize for yourself the main points from each paragraph. You can write these little summaries in the margins.
o Last but not least, you are very welcome to come to my office hours or to contact me if you have any questions.
Assessment methods
Decision whether students pass the practical will be calculated based on following performance indicators:
• 5 short tests (each of the tests constitutes 4% of the overal performance). Some of the tests will have the form of a quiz during a lecture, others will be homework's.
• Two research projects (each constitutes 25% of the overal performance). The projects will introduce students to (1) determination of protein structure by X-ray crystallography and (2) calculation of three-dimensional reconstruction from cryo-electron microscopy images. Both research projects will be due at 10:30am on the day listed in the Course outline section of this syllabus. Taking off 1% for each day the assignment is late will penalize late work. The research projects can be submitted only in electronic form.
• Active articipation in the practical (30% of the overal performance). Activity of students during the practical (asking questions and contributing to discussions).
Language of instruction
English
Further Comments
The course is taught once in two years.
The course is also listed under the following terms Autumn 2014, Autumn 2015.
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