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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


  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

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