期刊
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES
卷 369, 期 1647, 页码 -出版社
ROYAL SOC
DOI: 10.1098/rstb.2013.0322
关键词
femtosecond diffraction; crystallography; crystal injector; sample delivery; transmission electron microscopy (TEM); crystal characterization
类别
资金
- LCLS Ultrafast Science Instruments (LUSI) project - US Department of Energy, Office of Basic Energy Sciences
- DOE Office of Biological and Environmental Research
- National Institutes of Health, National Institute of General Medical Sciences [P41GM103393]
Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.
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