Biocrystallography (2009/2010)

Course partially running

Course code
4S00160
Credits
6
Coordinator
Ugo Luigi Monaco
Teaching is organised as follows:
Unit Credits Academic sector Period Academic staff
TEORIA 5 BIO/11-MOLECULAR BIOLOGY 2nd Semester Ugo Luigi Monaco
LABORATORIO 1 BIO/11-MOLECULAR BIOLOGY 2nd Semester Massimiliano Perduca

Learning outcomes

The goal of the Biocrystallography course for the degreee in Molecular and Industrial Biotechnology is to develop in the student the skills necessary to critically read and assess scientific papers in this branch of science which is central in modern Structural Biology.
After an introduction to the fundamentals of the theory of diffraction and the modern methods of data collection a detailed discussion of the phase problem is undertaken, along with the methods used to solve it for small molecules as well as macromolecules. During the course papers selected from the current literature dealing with important biological structures are read and discussed in detail. The laboratory introduces the student to the basic experimental techniques of the field, in particular those concerning the preparation of biological crystals.

Syllabus

THEORY

Introduction. Methods used to determine the three-dimensional structure of macromolecules. The role of Biocrystallography in macromolecular Structural Biology.

The theory of X-ray diffraction. Geometry of an X-ray scattering experiment. Scattering of a single electron and an atom. The atomic scattering factor. Structure factor. The structure factor of atoms not located at the origin. The diffraction pattern of a one-dimensional array of atoms. X-ray diffraction from a three-dimensional array of atoms. The von Laue scattering conditions. The structure factor of a crystal. Fourier transforms. Convolutions and their use in the computation of structure factors. Bragg’s law of diffraction.

Properties of crystals. Symmetry. Symmetry elements. Space groups. Reciprocal lattice. Preparation of macromolecular crystals. Properties of protein crystals. The relationship between the crystal lattice and the reciprocal lattice. The Ewald sphere. Determination of the space group and of the number of molecules in the unit cell of a macromolecular crystal.

Determination of the molecular structure by X-ray crystallography. The phase problem. Steps in determining the structure of a macromolecule. X-ray sources. Data collection methods. Solving the phase problem. The method of multiple isomorphous replacement. The Patterson function. Treatment of errors. Computation of electron density maps. Molecular replacement. Other methods used to solve the phase problem.

Model building and refinement. Interpretation of the electron density maps. Building the model. Refinement methods. Assessing the model quality. The R factor. Ramachandran plots. Checking the stereochemistry.

Some important results of Biocrystallography. Using the Fourier difference synthesis to study the function of proteins. Conformational changes. Time resolved Biocrystallography. The importance of synchrotron radiation.

Recommended Textbooks

!) Rupp, B. (2010) Biomolecular Crystallography. Garland Science, New York

2) Giacovazzo, C. (Editor) (2000) Fundamentals of Crystallography. Second Edition Oxford University Press, Oxford.

3) Blow, D.( 2005) Outline of Crystallography for Biologists Oxford University Press, Oxford.

4) Blundell, T. L. & Johnson, L. N. (1976) Protein Crystallography. Academic Press, New York.

5) Drenth, J (1994) Principles of Protein X-ray Crystallography. Springer Verlag.

6) Cantor, C. R. & Schimmel, P. R. (1980) Biophysical Chemistry Volume 2. W. H. Freeman and Company, San Francisco.

7) McPherson, A. (1983) The Preparation and Analysis of Protein Crystals. John Wiley, New York.

LABORATORY

The Biocrystallography practical lessons entail a series of meetings focused on the following arguments:
1) An introduction covering macromolecular crystallization theory and the most used techniques.
2) Preparation of protein crystals using Microdialysis, Hanging and Sitting drop.
3) Collection of a dataset with a rotating anode conventional source, an image plate detector and a low temperature cooling system.
4) Data processing and analysis.
5) Solution of a protein structure using molecular replacement techniques.
6) Protein model building and refinement.
7) Presentation of the most widely used programs for molecular graphics.

Assessment methods and criteria

oral

Reference books
Author Title Publisher Year ISBN Note
Bernhard Rupp Biomolecular Crystallography Garland Science 2009 978-0-8153-4081-2

Studying