Abstract
The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiationsensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-A resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.
| Original language | English |
|---|---|
| Pages (from-to) | 17122-17127 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 111 |
| Issue number | 48 |
| DOIs | |
| State | Published - Dec 2 2014 |
| Externally published | Yes |
Funding
Portions of this research were carried out at the Linac Coherent Light Source (LCLS), a National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The XPP and CXI instruments were funded through the LCLS Ultrafast Science Instruments project funded by the US Department of Energy, Office of Basic Energy Sciences. Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy Office of Biological and Environmental Research, and by the National Institutes of Health (NIH), National Institute of General Medical Sciences (including P41GM103393). A.T.B., A.Y.L., M.U., O.B.Z., and W.I.W. were supported in part by a Howard Hughes Medical Institute Collaborative Innovation Award that also provided funds for the purchase of the microdiffractometer. N.K.S. acknowledges the support of NIH GrantsGM095887 and GM102520 for data-processing methods. J.M.H. was supported by NIH Grants GM073210, GM082250, and GM094625. This work is supported as a part of the Biological and Electron Transfer and Catalysis EFRC, an Energy Frontiers Research Center funded by the US Department of Energy, Office of Science (DE-SC0012518).
Keywords
- Crystallography
- Femtosecond diffraction
- Structural biology
- XFEL