From electron microscopy to X-ray crystallography: molecular-replacement case studies

Abstract

Multi-component molecular complexes are increasingly being tackled by structural biology, bringing X-ray crystallography into the purview of electron-microscopy (EM) studies. X-ray crystallography can utilize a low-resolution EM map for structure determination followed by phase extension to high resolution. Test studies have been conducted on five crystal structures of large molecular assemblies, in which EM maps are used as models for structure solution by molecular replacement (MR) using various standard MR packages such as AMoRe, MOLREP and Phaser. The results demonstrate that EM maps are viable models for molecular replacement. Possible difficulties in data analysis, such as the effects of the EM magnification error, and the effect of MR positional/rotational errors on phase extension are discussed.

Figure 1

Effect of EM map magnification errors on the MR solution. (a) The vertical axis shows the gap in Patterson correlation separating the correct solution and the next highest noise peak using the program MOLREP. A minimum of 4 (yellow line) is the cutoff for a correct solution. The broken vertical line shows the optimal magnification of the EM map for the best MR solution. (b) Tests repeated using the program Phaser with the Z score shown on the vertical axis.

Figure 2

MR solution in the P2₁ crystal form. (a) The EM map model. The blue skeleton outlines the EM image. (b) The self-rotation plots from the X-ray data (left) and from the MR solution (right). (c) Comparison of the MR solutions from the EM map model (blue skeleton) and from the atomic model (yellow). The arrow points to the positional errors in the solution with the EM model.

Figure 3

Self-rotation (a) and packing analysis (b) of the P4₃2₁2 FAS crystal. The threefold axis of the FAS particle is indicated by the white triangle in (b).

Figure 4

Phase extension from the starting FAS EM map (a) to the final map at 4 Å (b).