"From Data to Structures" Program for the practical "Structure calculation and validation"

BNRA course 2004 

"From Data to Structures"
Program for the practical 
"Structure calculation and validation"

 

Chris Spronk and Aart Nederveen, May 2004

 

1. General remarks

The intention of the practical program of the 2004 BNRA course "From data to structures" is to make you familiar with the basics of NMR structure calculation and validation. Based on our previous experiences in giving this practical course, we have chosen to lead you through the coarse trajectory that one would follow when working on a biological macromolecule (see below). This means that a lot of the details of structure calculation will be skipped and some of the software will be used as a "black box". Our aim is that from the basic knowledge that is learned throughout the course, you will be able to set up your own structure calculations, and independently expand your knowledge on this topic to higher levels.
In practice, this means that instead of running many variations of different types of structure calculation protocols, a simple "standard" structure calculation protocol based on NOE-derived distance restraints and J-coupling-derived dihedral angle restraints will be performed. The calculated structures will then be analysed in detail using various structure validation methods and visual inspection. If time allows, it is possible to expand the practical to run structure calculations with other types of restraints and/or structure calculation protocols.
 

2. Setup

In order to reflect the reality of an NMR structure determination to some extent (and to allow for some variation in the final presentations), we have chosen to take a project -based approach. This means that each group will be assigned to calculate a set of structure models for a given protein. From these structures you will select "good" structures, investigate their global and local properties, identify possible problems in the structure etcetera. Following the detailed analysis of the structures, the focus shifts towards the biological relevance of the protein. This means, you will identify the regions in the protein structure that are of main biological interest, such as binding sites, active sites and so on (if present of course), and using the available literature and structures from the Protein Data Bank (PDB) to draw conclusions. You will present, compare and discuss your findings in a final presentation at the end of the course.

For each project the following is provided:

The deposited pdb-file
NMR distance and dihedral angle restraints in XPLOR/CNS format
The original paper describing the structure in pdf-format

The general goals for each project are:

Calculate and refine a structure ensemble using CNS
Analyse the structural properties and general quality of the molecule
Compare to the results described in the literature
Prepare a final presentation of the results

3. Software
3.1 Structure calculation

A large number of programs and protocols can be used to calculate macromolecular structures from NMR data. In this practical we will however concentrate on the use of one particular program, CNS ( "Crystallographic and NMR System") from Axel Br眉nger's laboratory. CNS is the follow-up of the widely used program X-PLOR. More information on both programs can be found at through the links at  .

3.2 Structure validation

Various different programs are available for structure validation. In this course we will use the two most powerful and widely used packages PROCHECK and WHAT IF. PROCHECK provides nice illustrative outputs, WHAT IF is more powerful but less user-friendly. Throughtout the course you will be made familiar with the strengths of both programs.

PROCHECK - Single structure analysis
PROCHECK-NMR - Structure ensemble analysis
PROCHECK-COMP - Comparison of two structures
WHAT IF - Single structure and/or ensemble analysis ( help pages )

3.3 Structure visualization

Throughout this course we plan to use molmol as a viewer. Maybe you are already familiar with this program. If not, take some time during this course to read the tutorial on the web. Several visualization tools that might be interesting for you are listed below.
PyMol - Powerful viewer ( tutorial )
Molmol - Powerful viewer and analysis program, but not very intuitive in its use (manual tutorial )
Rasmol - A simple and easy to use viewer, nice for quick inspection but rather limited in its capabilities ( tutorial )
Yasara   - Not used in this practical, but it is an extremely powerful and user friendly viewer and therefore worth mentioning, all you need is a good graphics card...)

 

4. Literature

The following papers are good overviews and starting points for reading:

  • P. Guentert. Structure calculation of biological macromolecules from NMR data. Quarterly reviews of biophysics 31, 145-237, (1998).
  • C.A.E.M. Spronk , S.B. Nabuurs, E. Krieger, G. Vriend & G.W. Vuister. Validation of protein structures derived by NMR spectroscopy. Progress in NMR spectroscopy, in preparation.
  • S.B. Nabuurs, C.A.E.M. Spronk , G. Vriend & G.W. Vuister. Concepts and tools for NMR restraint analysis and validation. Concepts in Magnetic resonance , accepted .