Diffraction & Diffusion des rayons X

Conceptual aspects

9h : Principles of X-ray scattering by macromolecules - Dominique Housset (IBS, Grenoble)

9h45: Practical aspects of Macromolecular X-ray crystallography - Patrice Gouet (IBCP, Lyon)

10h45: Small angle Xray scattering: a way to study the conformation, assemblies and interactions of biological macromolecules in solution - Dominique Durand (I2BC, Orsay)

11h30: SAXS versus Crystallography: the information you can get and respective limits - Marie-Hélène Le DU (CEA, Saclay)

Practical aspects

Softwares to be installed on your computer:

Instructions to download and install softwares for X-ray Macromolecule Crystallography (MX) and Small Angle X-ray Scattering (SAXS) used during the practical.

I - Small Angle X-ray Scattering (about 1h)

The goal of this practical is to analyse SAXS data from two amylosucrases, one from Neisseria polysaccharea (ASNP) and one from Deinococcus geothermalis (ASDG), and to show how the SAXS technique can help to determine the oligomeric state of these enzymes in solution.

Data and tutorials to dowload: SAXS Data (zip archive) & Tutorial (pdf format)

II - Macromolecular crystallograpy: the lysozyme as a case study (about 3h)

The goal of this practical is to show all the steps from diffraction images up to the electron density maps and model building, using some of the most popular sofwares in macromolecular crystallograpy.

During this practical course, you will be divided into three groups: Beginners. Medium. Advanced.

All groups will have at least to treat one set of x-ray diffraction data collected from a hen lysozyme crystal and solve the related crystal structure: chose your group according to your knowledge in x-ray crystallography.

Beginners 1/ iMosflm or XDS (depending on your computer OS) to process X-ray diffraction data and obtain observed amplitudes (Fobs); 2/ Molrep to solve the phase problem by the molecular replacement method; 3/ Refmac to refine the crystal structure; 4/ Coot to observe the final structure and the electron density maps and start model building.

Medium will use: 1/ iMosflm or XDS (depending on your computer OS) to process X-ray diffraction data of both native and gadolinium derivative lysozyme crystals; 2/ Phaser SAD pipeline to solve the phase problem by the SAD method; 2/ Molrep to solve the phase problem by the molecular replacement method; 4/ Refmacs to refine the crystal structures; 5/ Coot to observe the final structure and the electron density maps and start model building.

Advanced will use: 1/ iMosflm and XDS to process X-ray diffraction data of both native and gadolinium derivative lysozyme crystals; 2/ Molrep to solve the phase problem by the molecular replacement method; 3/ Phaser SAD pipeline to solve the phase problem by the SAD method; 4/ Refmacs to refine the crystal structures; 5/ Coot to observe the final structure and the electron density maps and start model building.

Data and tutorials to dowload:

- Crystallographic data for the practical (link to download a compressed (bzip2) tar archive, 2.69 Go)

a) Tutorial for Crystallographic data processing with XDS and commented input and output files: XDS.INP, IDXREF.LP, CORRECT.LP

b) Tutorial for Crystallographic data processing with iMosflm (CCP4)

c) Tutorial for Phasing by Single Anomalous Dispersion (SAD) and automated model building with SHELX, PHASER, PARROT, Buccaneer (pipeline CCP4) using Gd-derivative lysozyme data

d) Tutorial for Phasing by molecular replacement with Molrep (CCP4), refinement with Refmac (CCP4) using native lysozyme data and use of Coot for model building