Building and refining protein models within cryo-electron microscopy density maps based on homology modeling and multiscale structure refinement

Abstract

Automatic modeling methods using cryoelectron microscopy (cryoEM) density maps as constraints are promising approaches to building atomic models of individual proteins or protein domains. However, their application to large macromolecular assemblies has not been possible largely due to computational limitations inherent to such unsupervised methods. Here we describe a new method, EM-IMO (electron microscopy-iterative modular optimization), for building, modifying and refining local structures of protein models using cryoEM maps as a constraint. As a supervised refinement method, EM-IMO allows users to specify parameters derived from inspections so as to guide, and as a consequence, significantly speed up the refinement. An EM-IMO-based refinement protocol is first benchmarked on a data set of 50 homology models using simulated density maps. A multiscale refinement strategy that combines EM-IMO-based and molecular dynamics-based refinement is then applied to build backbone models for the seven conformers of the five capsid proteins in our near-atomic-resolution cryoEM map of the grass carp reovirus virion, a member of the Aquareovirus genus of the Reoviridae family. The refined models allow us to reconstruct a backbone model of the entire grass carp reovirus capsid and provide valuable functional insights that are described in the accompanying publication [Cheng, L., Zhu, J., Hui, W. H., Zhang, X., Honig, B., Fang, Q. & Zhou, Z. H. (2010). Backbone model of an aquareovirus virion by cryo-electron microscopy and bioinformatics. J. Mol. Biol. (this issue). doi:10.1016/j.jmb.2009.12.027.]. Our study demonstrates that the integrated use of homology modeling and a multiscale refinement protocol that combines supervised and automated structure refinement offers a practical strategy for building atomic models based on medium- to high-resolution cryoEM density maps.

Figure 1

Performance of EM-IMO-based refinement protocol on 50 CASP proteins using simulated density maps. (a) Backbone RMSD (N, Cα and C) of the homology model with respect to the experimental structure before (in black) and after (in grey) one iteration of refinement using the EM-IMO-based protocol. (b) Density fitting score of the homology model before (in black) and after (in grey) refinement. (C) Distribution of 371 regions against the RMSD change during refinement.

Figure 2

Two examples of EM-IMO refinement from the CASP data set using density maps simulated at 7 Å. (a) C-terminus of T0132 (125-151). (b) Segment of T0298 (159-197) with a missing helix (165-179). The initial model is shown in magenta, while the refined region is shown in blue and the native structure in green.

Figure 3

Near-atomic resolution cryoEM map of GCRV virion and the seven refined backbone models of all five GCRV structural proteins present in the virion. (a) Protein structures of the core. Five distinct structures/conformers of the three core proteins (two conformers of VP3: VP3A and VP3B; two conformers of the clamping protein VP6: VP6A and VP6B; and turret protein VP1) exist in inner capsid core. The cryoEM density is shown in semi-transparently gray and the final refined model is shown in ribbon. The full model of the core is shown in the center with each of the seven unique protein structures shown in a different color. (b) Proteins of the outer shell. The outer shell is made up of trimers of VP5/VP7 dimer. On the left two panels are the cryoEM density maps of VP7 and VP5 (semi-transparently gray) superimposed with the final refined models shown in ribbon. Top and side views of the ribbon model of the trimer are shown in the middle. The full model of the outer shell is shown at the right with each of the seven unique protein structures shown in a different color.

Figure 4

Refinement of the VP1 C-terminal domain (1128-1299). (a) Initial homology model. (b) Homology model after a short EM-IMO refinement of the head domain. In a and b the protein body is shown in magenta and the C-terminal domain in cyan. Two inserted figures show the EM-IMO refinement for a loop region (46-57) and a double-helical region (896-926), with the refined structure in green.

Figure 5

Refinement of the VP5 head domain (287-473). Initial homology model (up-left corner) is in magenta and the head domain is marked in cyan. Two inserted figures on the right show the head domain after a single EM-IMO refinement (in orange) and after the subsequent 5 iterations of refinement using the EM-IMO-based protocol (in red). Two inserted figures on the bottom show the EM-IMO refinement for loop regions 222-236 and 549-559, with the refined structure in green.

Figure 6

Refinement of the VP3B N-terminus. (a) λ1B structure (green) in the three-fold symmetry. (b) Initial homology model (yellow). (c) Homology model built based on the tuned sequence alignment (cyan). In b and c the template structure is shown in green and the key residues used in the alignment tuning are marked. (d) Final model after five iterations of refinement using the EM-IMO-based protocol (red), with the first 18 residues truncated.

Figure 7

Refinement of the VP7 model. (a) Sequence alignment of GCRV VP7 and MRV σ3. (b) Initial homology model (center, magenta) with four inserted figures showing regions subjected to the manual adjustment of sequence alignment and topology (yellow). (c) Homology model built after adjustment. (d) Final model after 5 iterations of refinement using the EM-IMO-based protocol.

Figure 8

MDFF refinement of GCRV proteins. (a) Plot of normalized cryoEM pseudo-energy (ref. 35) during 200-step energy minimization (b) Plot of normalized cryoEM pseudo-energy (ref. 35) during 1-ns MDFF simulation.

Figure 9

Flow charts of local and global refinement. (a) Flow chart of the EM-IMO program, which is a local refinement method. (b) Typical steps involved in refining full protein structure. Our procedure is basically a global refinement protocol using EM-IMO as a core component, thus termed EM-IMO-based refinement protocol.