PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Families in Gene Neighborhoods
June 2015
Expanding the Reach of SAD
April 2015
Greasing the Path for SFX
January 2015
Time-Resolved Crystallography with HATRX
December 2014
Structures Without Damage
August 2014
Error Prevention
July 2014
A Refined Refinement Strategy
May 2014
Membrane Proteome: Microcrystals Yield Big Data
April 2014
Optimizing Damage
February 2014
Getting Better at Low Resolution
January 2014
Building a Structural Library
November 2013
Drug Discovery: Identifying Dynamic Networks by CONTACT
October 2013
Microbiome: Solid-State NMR, Crystallized
September 2013
Fluorescence- and Chromatography-Based Protein Thermostability Assay
October 2012
Insert Here
October 2012
Native phasing
August 2012
Smaller may be better
April 2012
Metal mates
February 2012
Not so cool
December 2011
One from many
August 2011
Rosetta hone
July 2011
Solutions in the solution
June 2011
Beyond crystals, solutions, and powders
May 2011
Snapshot crystallography
March 2011
FERM-ly bound
February 2011
A new amphiphile for crystallizing membrane proteins
January 2011
'Super-resolution' large complexes
December 2010
Proteinase K and Digalacturonic Acid
September 2010
Some crystals like it hot
May 2010
Tips for crystallizing membrane proteins in lipidic mesophases
February 2010
Tackling the phase problem
November 2009
Crystallizing glycoproteins
September 2009
Crystals from recalcitrant proteins
August 2009
Tips for crystallizing membrane proteins
June 2009
Chaperone-assisted crystallography
March 2009
An “X-ray” ruler
January 2009
Methylation boosts protein crystallization
December 2008

Technology Topics Crystallography

Not so cool

SBKB [doi:10.1038/sbkb.2011.54]
Technical Highlight - December 2011
Short description: A comparison of X-ray diffraction datasets obtained at ambient and cryogenic temperatures reveals temperature-dependent effects on structural models.

Small packing voids (red spheres) and alternative sidechain conformations are shown for the protein lysozyme. Image courtesy of James Fraser.

Cryogenic cooling of protein crystals has become standard practice for structural biologists, and the benefits are hard to argue against. Flash-frozen crystals transport easily to a synchrotron and are less susceptible to radiation damage, contributing to high-resolution structures of thousands of proteins. It is widely thought that cooling the crystals has minimal effects on the structural models, but several studies have indicated this procedure may introduce biases including protein shrinkage and reductions in local motions.

To re-examine this question, Alber and colleagues (PSI JCSG) compared structural models for 30 different proteins for which data were available at ambient and cryogenic temperatures. Using the programs qFit and Ringer to detect structural ensembles, the authors found that cryogenic cooling frequently remodels exposed and buried residues, increases lattice contact area, and eliminates certain functional micro-cavities. One of the major alterations is seen when comparing X-ray models with NMR data: in contrast to models from flash-frozen crystals, ambient-temperature crystallographic models show more conformations corresponding to flexible regions detected in solution using NMR. This was evident when examining X-ray and NMR data for the GTPase H-Ras. In H-Ras bound to a substrate analog, a catalytically incompetent conformation predominated electron density maps from both ambient and cryo-cooled conditions. However, at low electron density values often regarded as noise, the ambient-temperature data also displayed small populations of alternative conformations in segments previously found to be flexible in solution using NMR. These rare substates, including the catalytically competent conformation, were absent from the cyrogenic crystallographic model.

The authors propose that models from cryo-cooled crystals represent low energy, low entropy states that may be missing relevant alternative conformations. While these conformations can be discovered using Ringer or qFit on cryogenic data, ambient temperature data contain more information regarding conformational ensembles. As a result, they suggest that efforts be directed to ambient temperature data collection with emphasis on optimizing techniques that minimize radiation damage. This approach, coupled with new computational methods to define alternative conformations, would lead to structural models that are more informative and more representative of proteins in vivo.

Steve Mason

References

  1. J. Fraser et al. Accessing protein conformational ensembles using room-temperature X-ray crystallography.
    Proc Natl Acad Sci U S A. 108, 16247-16252 (2011). doi:10.1073/pnas.1111325108

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health