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Abstract
X-ray crystallography and cryo-electron microscopy (cryo-EM) are complementary techniques for structure determination. Crystallography usually reveals more detailed information, while cryo-EM is an extremely useful technique for studying large-sized macromolecules. As the gap between the resolution of crystallography and cryo-EM data narrows, the cryo-EM map of a macromolecule could serve as an initial model to solve the phase problem of crystal diffraction for high-resolution structure determination. FSEARCH is a procedure to utilize the low-resolution molecular shape for crystallographic phasing. The IPCAS (Iterative Protein Crystal structure Automatic Solution) pipeline is an automatic direct-methods-aided dual-space iterative phasing and model-building procedure. When only an electron-density map is available as the starting point, IPCAS is capable of generating a completed model from the phases of the input map automatically, without the requirement of an initial model. In this study, a hybrid method integrating X-ray crystallography with cryo-EM to help with structure determination is presented. With a cryo-EM map as the starting point, the workflow of the method involves three steps. (1) Cryo-EM map replacement: FSEARCH is utilized to find the correct translation and orientation of the cryo-EM map in the crystallographic unit cell and generates the initial low-resolution map. (2) Phase extension: the phases calculated from the correctly placed cryo-EM map are extended to high-resolution X-ray data by non-crystallographic symmetry averaging with phenix.resolve. (3) Model building: IPCAS is used to generate an initial model using the phase-extended map and perform model completion by iteration. Four cases (the lowest cryo-EM map resolution being 6.9 Å) have been tested for the general applicability of the hybrid method, and almost complete models have been generated for all test cases with reasonable Rwork/Rfree. The hybrid method therefore provides an automated tool for X-ray structure determination using a cryo-EM map as the starting point.
Highlights
X-ray crystallography has played a fundamental role in the field of structural biology to provide a mechanistic understanding of critical biological processes
If extremely high-resolution X-ray In 2015, we demonstrated that starting with a partial model data are available, one such method is ab initio phasing, as that is as low as 30% of the protein complex, IPCAS is capable implemented in ARCIMBOLDO (Rodrıguez et al, 2009). of extending the starting structure generated from MR to an Initial phases can be derived experimentally from almost complete complex structure with reasonable Rwork and isomorphous or anomalous differences using heavy-atom Rfree values (Zhang et al, 2015)
The input data are first passed through a model-building and refinement process implemented in Phenix.AutoBuild to derive an initial model, and the resultant model is used as the starting point for directmethods-aided model completion by iteration, including real
Summary
X-ray crystallography has played a fundamental role in the field of structural biology to provide a mechanistic understanding of critical biological processes. Of extending the starting structure generated from MR to an Initial phases can be derived experimentally from almost complete complex structure with reasonable Rwork and isomorphous or anomalous differences using heavy-atom Rfree values (Zhang et al, 2015) This procedure integrates diffraction, or phases can be obtained using molecular several programs and can call these individual programs in replacement (MR) when suitable models for placement in the three parts of its workflow: (1) reciprocal-space phase unit cell are known. The initial phases from the map replacement are extended to high resolution X-ray diffraction data by iterated density modification implemented in the program Phenix v.1.12-2829 (the RESOLVE density modification subroutine) (Terwilliger, 2002). This strategy is rather powerful when there is a high degree of internal symmetry or sufficient resolution
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