Abstract
Structures of multisubunit macromolecular machines are primarily determined either by electron microscopy (EM) or by X-ray crystallography. In many cases, a structure for a complex can be obtained at low resolution (at a coarse level of detail) with EM and at a higher resolution (with finer detail) by X-ray crystallography. The integration of these two structural techniques is becoming increasingly important for the generation of atomic models of macromolecular complexes. A low-resolution EM image can be a powerful tool for obtaining the 'phase' information that is missing from an X-ray crystallography experiment; however, integration of EM and X-ray diffraction data has been technically challenging. Here we present a step-by-step protocol that explains how low-resolution EM maps can be placed in the crystallographic unit cell by molecular replacement, and how initial phases computed from the placed EM density are extended to high resolution by averaging maps over noncrystallographic symmetry. As the resolution gap between EM and X-ray crystallography continues to narrow, the use of EM maps to help with X-ray crystal structure determination, as described in this protocol, will become increasingly effective.
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Acknowledgements
R.N.J. is supported by the National Research Service Award postdoctoral fellowship (F32 GM108436) from the US National Institutes of Health (NIH). R.J.R. and T.C.T. are supported by a grant (GM063210) from the NIH. R.J.R. is supported by a Principal Research Fellowship from the Wellcome Trust (grant no. 082961/Z/07/Z). Research in the Wiedenheft lab is supported by the NIH IDeA Program COBRE (GM110732), an R01 to B.W. (GM108888), the National Science Foundation EPSCoR (EPS-110134), the M.J. Murdock Charitable Trust and the Montana State University Agricultural Experimental Station.
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All authors contributed to writing the protocol. A.J.M. developed the EM refinement parameter in Phaser-MR necessary to account for uncertainty in the EM magnification error.
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Jackson, R., McCoy, A., Terwilliger, T. et al. X-ray structure determination using low-resolution electron microscopy maps for molecular replacement. Nat Protoc 10, 1275–1284 (2015). https://doi.org/10.1038/nprot.2015.069
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DOI: https://doi.org/10.1038/nprot.2015.069
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