Journal
IUCRJ
Volume 2, Issue -, Pages 421-430Publisher
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S2052252515009811
Keywords
serial femtosecond crystallography; viscous crystal delivery; protein complexes; membrane proteins; femtosecond studies; nanocrystals; coherent X-ray diffractive imaging; free-electron laser
Funding
- STC Program of the National Science Foundation through BioXFEL [1231306]
- National Institutes of Health Femtosecond Nano-crystallography of Membrane Proteins Award [617095583]
- Centre for Applied Structure Discovery
- NIH [U54GM094599]
- NIGMS [R01 GM108635]
- DOE [DE-SC0010575, GM097463]
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]
- U.S. Department of Energy (DOE) [DE-SC0010575] Funding Source: U.S. Department of Energy (DOE)
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Serial femtosecond crystallography (SFX) has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4 degrees C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 angstrom resolution using 300 mu g of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.
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