Journal
NATURE PROTOCOLS
Volume 10, Issue 9, Pages 1275-1284Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2015.069
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Funding
- National Research Service Award postdoctoral fellowship from the US National Institutes of Health (NIH) [F32 GM108436]
- NIH [GM063210]
- Principal Research Fellowship from the Wellcome Trust [082961/Z/07/Z]
- National Science Foundation EPSCoR [EPS-110134]
- M.J. Murdock Charitable Trust
- Montana State University Agricultural Experimental Station
- NIH IDeA Program COBRE, an R01 [GM110732, GM108888]
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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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