Erice2010:workshop6

This is the detailed information for workshop #6, Analysis of large assemblies, led by Dima Chirgadze

Aim

Studies of large proteins or multiprotein assembles remain a challenge to structural biologists, who are likely to be confronted with complex problems at every stage of the crystallographic roadmap leading to 3D structure, i.e. at production of protein or complexes, crystallisation and structure solution. The sheer size of such objects is one of the main contributors to the complexity of the task, often resulting in low-resolution diffraction data and difficulties during the structure determination calculations.

As far as crystallographic calculations are concerned there are two common scenarios when attempting to determine the structures of large assemblies. The first of these is when some or all of the components – protomers or even domains of multidomain components – of the multiprotein assembly are of known 3D structure. This allows a “divide-and-conquer” approach, i.e. finding the positions of individual components using the Molecular Replacement, which can be combined with real-space search procedures in difficult cases. The second scenario involves a large protein or obligate complex of unknown structure for which a standard “de-novo” structure determination process using the plethora of experimental methods (MIR, SAD, MAD, SIRAS, MIRAS) for obtaining the initial phases must be utilized.

The aim of this workshop is to introduce the participant to some particularities of the process of structure solution of large protein assemblies by X-ray crystallography using “real-life” examples. In particular, it demonstrates how to perform a customized multicomponent Molecular Replacement search, real space fitting procedure (Thyroid-Stimulating Hormone Receptor/M22Fab autoantibody complex) as well as giving an example of experimental low-resolution electron density maps and a manual model building into those maps (DNA-dependent Protein Kinase catalytic subunit in complex with C-terminal domain of Ku80).

Suitability

The workshop is suitable for novice and intermediate users. Basic knowledge of unix shell scripting commands and methods in protein crystallography are recommended. If there are advanced areas that people are interested in discussing, we can meet informally during the Erice workshop.

Materials

The following articles give some background information on the “real-life” examples used in this workshop:

• Sanders, J., Chirgadze, D.Y., Sanders, P., Baker, S., Sullivan, A., Bhardwaja, A., Bolton, J., Reeve, M., Nakatake, N., Evans, M., Richards, T., Powell, M., Nunez, R.M., Blundell, T.L., Furmaniak, J. and Rees-Smith, B. (2007) Crystal structure of the TSH receptor in complex with a thyroid stimulating autoantibody. Thyroid, 17, 395-410.
• Sibanda, B.L., Chirgadze, D.Y. and Blundell, T.L. (2010) Crystal structure of DNA-PKcs reveals a large open-ring cradle comprised of HEAT repeats. Nature, 463,118-121.

See also the following documentation on the programs used in the workshop:

• AMoRe (CCP4: supported program)
• FFFear (CCP4: supported program)
• Coot

Protocol

This is a “watch me” style workshop, so you will not excepted to do anything at all. However, the example data files and scripts will be available for download as well as the workshop presentation, so you would be able to go through the workshop yourself.

The files are available here.

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