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

Structures Without Damage

SBKB [doi:10.1038/sbkb.2014.215]
Technical Highlight - August 2014
Short description: Radiation damage-free protein structures can be solved using an X-ray free-electron laser and large single crystals.

Schematic of approach showing (a) combined usage of a large crystal and femtosecond XFEL pulses and (b) variation in the number of diffraction spots along the vertical direction.

One of the facts that protein crystallographers must face is radiation damage: the X-ray beam generates reactive species that attack the crystal, often causing damage before a full diffraction dataset can be collected.

The ultrashort, femtosecond pulses at X-ray free-electron laser (XFEL) facilities offer a way around this problem: datasets can be collected before the radiation damage destroys the crystal. This has mainly been accomplished using serial femtosecond crystallography. In this approach, a large number of microcrystals are streamed into the path of the XFEL beam, and a single diffraction snapshot is taken of each microcrystal before it is destroyed. Although serial femtosecond crystallography has proven to be very powerful and has resulted in the solution of novel, exciting structures, the approach is still fraught with a number of technical challenges that limit its applicability.

The use of larger single crystals, on the other hand, allows the collection of a series of diffraction snapshots as the crystal is rotated. Using large crystals can also ensure high-quality data, simplifying subsequent data analysis as compared to the serial approach.

Ago, Yoshikawa and colleagues implemented this new method at the SPring-8 angstrom compact free-electron laser facility in Japan, to solve the structure of bovine heart cytochrome c oxidase (CcO). The structure of this metalloenzyme was previously solved by traditional synchrotron X-ray diffraction, but the geometry of its peroxide ligand in the oxidized state could not be resolved due to peroxide reduction caused by radiation damage.

The authors collected 1,396 still diffraction images from 76 individual, large CcO crystals that were rotated in 0.1° increments. They merged the data and solved the 1.9-Å structure (PDB 3WG7) using traditional crystallography software tools and, for the first time, were able to observe the pose of the peroxide ligand in the fully oxidized state. The approach may prove to be as useful in solving the structures of other highly radiation-sensitive metalloproteins.

Allison Doerr

References

  1. K. Hirata et al. Determination of damage-free crystal structure of an X-ray-sensitive protein using an XFEL.
    Nat. Methods. 11, 734-736 (2014). doi:10.1038/nmeth.2962

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