From lows to highs: using low-resolution models to phase X-ray data

Abstract

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an $13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.

How cryo‐electron microscopy and X‐ray crystallography complement each other

“…In phasing the high‐resolution X‐ray data with low‐resolution EM reconstruction by molecular replacement, once the search EM map has been positioned, theoretical phases can be calculated up to the EM reconstruction resolution. This has been used in determining the structures of some large complexes such as viruses, ribosomes, and others . A few years ago, in order to generate enough overlap between the low resolution of cryo‐EM map and the crystallographic diffraction data, a greater camera length or a smaller beam blocker had to be used to collect low frequency signals …”

“…This has been used in determining the structures of some large complexes such as viruses, ribosomes, and others. 21,22 A few years ago, in order to generate enough overlap between the low resolution of cryo-EM map and the crystallographic diffraction data, a greater camera length or a smaller beam blocker had to be used to collect low frequency signals. 20 The current progress of single particle cryo-EM reconstruction allows reconstruction of relatively small molecules at sub-nanometer resolution.…”

Iterative projection algorithms in protein crystallography. II. Application

“…Alternatively, if a low-resolution envelope for the oligomer was generated experimentally using small-angle X-ray solution scattering (SAXS) or transmission electron microscopy (TEM), it might be positioned within the unit cell. There is a precedent for using low-resolution TEM-derived image reconstructions and SAXS-derived envelopes as search models in molecular replacement (Urzhumtsev & Podjarny, 1995;Dodson, 2001;Hao, 2006;Navaza, 2008;Xiong, 2008;Hong & Hao, 2009;Trapani et al, 2010;Stuart & Abrescia, 2013). In many cases the orientation of the oligomer within the crystal can be deduced from inspection of the self-rotation function (Tong & Rossmann, 1997;Sawaya, 2007), providing an independent check on the validity of any solution (Dodson, 2001;Xiong, 2008).…”

Single crystal X-ray diffraction analysis of virus structure and its applications in the development of pharmaceutical agents

“…[31] Initially, cryoEM techniques were used in conjunction with virus crystallography as a method to generate low-resolution models of viruses for phasing the X-ray diffraction data of virus crystals. [29,[32][33][34] The phases were then extended to high resolution step-by-step based on electron density averaging as described in Section 2. As more structure determination results by density averaging were reported, it became unnecessary in many cases to use the low resolution cryoEM structure as the initial phasing model.…”