title: X-ray structure determination using low-resolution electron microscopy maps for molecular replacement.
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.
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Jackson
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK.
authors.forename: Airlie J
authors.initials: AJ
authors.lastname: McCoy
authors.affiliation: Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
authors.forename: Thomas C
authors.identifier: 0000000163840320
authors.initials: TC
authors.lastname: Terwilliger
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK.
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Read
authors.affiliation: Department of Microbiology and Immunology, Montana State University, Bozeman, Montana, USA.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
mesh_terms: D064130:CRISPR-Associated Proteins; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D008854:Microscopy, Electron; D015394:Molecular Structure
chemical_list: D064130:CRISPR-Associated Proteins