PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons

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

Native phasing

SBKB [doi:10.1038/sbkb.2011.94]
Technical Highlight - August 2012
Short description: Anomalous diffraction from multiple crystals allows phasing of data from native proteins.

Ribbon representation of the netrin G2 structure obtained using phase information acquired from native sulfur atoms (magenta spheres) and a calcium ion (red sphere). Figure courtesy of Wayne Hendrickson.

Solving the crystal structure of a new protein without prior structural information on closely related proteins requires a solution to the phase problem. Since the advent of multi- and single-wavelength anomalous diffraction (MAD and SAD) experiments many new structures have been solved once the necessary selenomethionyl or heavy atom sites were incorporated. While MAD and SAD experiments are now very widely used to acquire phases there remain problematic cases where appropriate heavy atoms cannot be introduced readily and are not intrinsically present.

Hendrickson and colleagues (PSI NYCOMPS) have investigated the possibility of routinely obtaining phases using anomalous diffraction from lighter atoms (notably sulfur and phosphorous) intrinsically present in native macromolecules. The underlying technique is not new, but the success of experiments on selenomethionyl proteins combined with the weakness of anomalous scattering signals from intrinsic sites have limited application mainly to test cases. The key solution is to achieve data multiplicity while avoiding radiation damage and other sources of error.

The authors extended their earlier work to develop a robust native SAD method for enhancing weak signals by combining data from multiple crystals. They optimized the x-ray energy and beam path parameters by minimizing background absorption and matching the beam size to crystal size. To address concerns about crystal variation, for example as introduced by freezing, the authors used cluster analyses to check the statistical equivalence and integrity of the crystals used. The method was applied to four proteins, three of which (HK9S, CysZ and netrin G2) yielded new structures while the fourth (TorT/TorSS) is a complex with a known structure, but was challenging due to its size and complexity. The resulting structures used 5 to 13 crystals each and were solved at resolutions of 2.3 to 2.8 Å.

Although the method was successful as implemented, the authors highlight ways to improve both data collection and analysis. They suggest that an advanced undulator beamline optimized for low-energy x-ray experiments and a high-angle detector could be combined with improved data handling. While the method requires multiple crystals, it is possible that benefits will accrue from further experiments on native phasing sites in both nucleic acids and proteins.

Michael A. Durney


  1. Q. Liu et al. Structures from anomalous diffraction of native biological macromolecules.
    Science 336, 1033-1037 (2012). doi:10.1126/science.1218753

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