Structural Insights from Membrane Small-Angle X-ray Diffraction with Anomalous X-ray Scattering
Anomalous X-ray scattering from lanthanide ions can probe the location of membrane protein Ca2+ sites in their role in protein function and intercellular communication. Despite the small size of these probes, their localization is possible even in diffraction experiments conducted at considerably lower resolution than the size of the lanthanide ion itself. Their electronic structure provides for large changes in their X-ray scattering power as a very sensitive function of the X-ray energy, and hence they enable an effective isomorphous replacement by only changing the incident X-ray energy by a few 10 of eVs where no other atom’s scattering power is changing. The feasibility of this experimental approach is facilitated by the harvesting of hard X-ray white light from synchrotron radiation and the development of monochromators able to select very narrow windows of X-ray energy. The resolution enhancement afforded by these ions and this experimental paradigm facilitates the localization of these Ca2+ binding sites.
KeywordsElectron Density Profile Iterative Refinement Anomalous Scattering National Synchrotron Light Source Calculated Amplitude
The authors gratefully acknowledge the team of people that have encouraged and supported the studies described here. Keith Hodgson recruited RHF to his lab as a post doc. Keith’s chemistry lab is inextricably tied to the SSRL synchrotron X-ray source as a result of the vision of Sebastian Doniach who reigned as the first director of the Stanford Synchrotron Radiation Project that morphed into SSRL. RHF’s introduction to membrane diffraction came in Bob Stroud’s amazing lab that generously opened to RHF where he learned that the operation of the Elliot X-ray generator was temperamentally tied to the care and love of Mel O. Jones. The small-angle camera existed there and provided RHF’s introduction to the membrane diffraction paradigm when Mel had the Elliot humming and RHF had mastered the AChR preparation from Torpedo electric organ at the mentoring of Michael Klymkowsky. David Agard, who wrote the constrained iterative refinement program, provided insight to its application to newly acquired data and was a constant inspiration for his enthusiastic approach to life and science. As the anomalous data was coming in, Janet Finer-Moore miraculously appeared to provide expert advice to RHF about the conversion of the crystallographic heavy atom refinement program to one suited for small-angle diffraction data instead of crystallographic spots. Meanwhile back at the synchrotron was a team of people who built the small-angle camera and prepared samples for irradiation including Rick Miake-Lye, Stevan Hubbard, and Soichi Wakatsuki, who were then joined for the weeks of all night partying collecting data 24/7 on the various additional samples prepared by Jeff Reidler, Jean Luc-Ranck, and RHF. God bless the dedicated runs’ overnight shifts at SSRL when the data is pouring in with a vengeance with the machinists banging new steel work stations into shape right next door in the SSRL workshop. RHF and TEL are also grateful for our long suffering wives who endured our enthusiasm for this work with very little reason for so doing. Thank you all.
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