Protein Structural Modeling for Electron Microscopy Maps Using VESPER and MAINMAST

Abstract

An increasing number of protein structures are determined by cryo-electron microscopy (cryo-EM) and stored in the Electron Microscopy Data Bank (EMDB). To interpret determined cryo-EM maps, several methods have been developed that model the tertiary structure of biomolecules, particularly proteins. Here we show how to use two such methods, VESPER and MAINMAST, which were developed in our group. VESPER is a method mainly for two purposes: fitting protein structure models into an EM map and aligning two EM maps locally or globally to capture their similarity. VESPER represents each EM map as a set of vectors pointing toward denser points. By considering matching the directions of vectors, in general, VESPER aligns maps better than conventional methods that only consider local densities of maps. MAINMAST is a de novo protein modeling tool designed for EM maps with resolution of 3-5 Å or better. MAINMAST builds a protein main chain directly from a density map by tracing dense points in an EM map and connecting them using a tree-graph structure. This article describes how to use these two tools using three illustrative modeling examples. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protein structure model fitting using VESPER Alternate Protocol: Atomic model fitting using VESPER web server Basic Protocol 2: Protein de novo modeling using MAINMAST.

Keywords: cryo-EM alignment; de novo protein modeling; electron microscopy maps; protein fitting; protein structure.

Figure 1

Overview of VESPER method. Steps of VESPER are shown on the left, and an example of using VESPER for aligning a pair of EM maps is shown on the right. The right panel shows the alignment of the complete V‐ATPase, which has EMD‐8724 determined at resolution 6.8 Å (the structure in pink on the left), and the Vo region of the V‐ATPase, which has EMD‐8409 determined at resolution 3.9 Å (the structure in cyan on the right). The unit vector representation of EMD‐8724 and EMD‐8409 are shown in red and dark blue, respectively.

Figure 2

Overview of MAINMAST. Steps of the MAINMAST algorithm are illustrated on the left, and an example of MAINMAST on building a protein model from a cryo‐EM map of structural protein 5 of cytoplasmic polyhedrosis virus solved at a 2.9 Å resolution (EMD‐6374) is shown on the right (modified figure taken from Figure 1 in the MAINMAST paper. The reuse is permitted under Creative Commons Attribution 4.0 International License).

Figure 3

Results of the VESPER program on EMD‐3342 and simulated map of PDB entry 5FWM. (A) Experimental map EMD‐3342, (B) EMD‐3342 in gray and the vector representation of the top model by VESPER of protein complex PDB: 5FWM. The top model is shown as a set of spheres of different colors, where blue means that the matched vectors of the two EM maps are aligned well, green means no alignment, and red represents the alignment in opposite directions. (C) EMD‐3342 in gray and the top model of VESPER in cyan, which has an RMSD of 3.44 Å.

Figure 4

Screenshot of the main page of the VESPER web server.

Figure 5

Screenshot of the SPD3 file generated by SPIDER2 for Chain A of PDB: 4CI0.

Figure 6

MAINMAST output of EMD‐2513, chain A. (A) The longest path of the MST i.e., path.pdb, (B) the LDPs generated by the mean shift clustering algorithm, (C) MST generated using tree mode in MAINMAST, (D) all edges using graph mode in MAINMAST.

Figure 7

The modeled protein structure by MAINMAST from EMD‐2513, chain A. (A) The native structure of 4CI0, chain A in yellow and the Cα model by MAINMAST in cyan, (B) the refined full‐atom modeled protein by MAINMAST in magenta and the native structure in yellow, (C) the native and modeled protein structures fitted to chain A segmented map of EMD‐2513.

Figure 8

Best fittings of 5FWM in EMD‐3342 by VESPER. (A) EMD‐3342 with its fitted native atomic structure, PDB entry: 5FWM, (B) the best model by the VESPER program which has RMSD of 3.44 Å, (C) the best model by the VESPER web server with RMSD of 5.8 Å.

Figure 9

Top 10 models of 5FWM in EMD‐3342 by the VESPER program. Native atomic structure, 5FWM is in yellow, EMD‐3343 in gray, and the top models numbered from 1 to 10 are in the other colors.

Figure 10

Modeling the entire structure of PDB entry, 4CI0 from EM map 2513 using VESPER and MAINMAST. (A) The native structure, 4CI0, in yellow and the best model by VESPER and MAINMAST in cyan; (B) the native and best structures fitted in EMD‐2513.