Structure Calculation and Validation

Introduction

Protein structure determination by nuclear magnetic resonance (NMR) spectroscopy is a burdgeoning field of study that encompasses a wide variety of techniques and methodologies.  In addition, validation of structures determined during and at the end of the structure determination process is critical to the accuracy of the final structures.  Under Structure Calculation and Validation we describe the standard protocols for protein structure determination adopted by the NMR laboratories in the NESG.  The section is broadly divided into 4 categories: 

• Structure Calculation
• Structure Refinement
• Special Topcis
• Structure Validation and Deposition

Structure Calculation

The Structure Calculation chapter features several sub-categories assigned on the basis of the program or approach used for protein structure calculation.  Here is a brief description of each sub-category:

CYANA and AutoStructure

CYANA and AutoStructure are the two primary programs used in the NESG for initial protein structure calculation.   CYANA is a torsion angle dynamics based approach and can be run with manually assigned NOEs and distance constraints or in fully automated NOESY assignment mode.  Structures are computed in several cycles and structures those the lowest target function are retained at the conclusion of each cycle.  AutoStructure uses a bottom-up approach and internal automated NOESYASSIGN module for iterative automated NOESY assignment.  In each cycle of AutoStructure, distance and torsion angle constraints are fed into either CYANA or XPLOR for structure calculation. Again, structures qith the lowest target function or energy are collected for the subsequent cycle of calculations.

For automated structure calculations, required input for each program includes: 

• protein sequence
• chemical shift assignment list
• NOESY peak lists
• torsional angle constraints (i.e., from TALOS)
• user defined options including:  total number of structure calculated, number of "best" structures kept at the end of each cycle.

Optional input for the programs includes:

• manual distance constraints
• hydrogen bond constraints
• AutoStructure can also interpret J-coupling and slow N-H exchange data in its initial secondary structure and fold analysis
• The newest version of CYANA (3.0) can use orientational constraints (i.e., residual dipolar couplings); CYANA is also preferable for dimer structure calculations
  

In general, central to the use of these programs is the complete or near complete assignment of resonances in the protein of interest as well as careful analysis and peak picking of NOESY (2D, 3D, 4D) spectra

Structure Validation and Quality Assessment

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