Current approaches for automated model building into cryo-EM maps using Buccaneer with CCP-EM

Abstract

This work focuses on the use of the existing protein-model-building software Buccaneer to provide structural interpretation of electron cryo-microscopy (cryo-EM) maps. Originally developed for application to X-ray crystallography, the necessary steps to optimise the usage of Buccaneer with cryo-EM maps are shown. This approach has been applied to the data sets of 208 cryo-EM maps with resolutions of better than 4 Å. The results obtained also show an evident improvement in the sequencing step when the initial reference map and model used for crystallographic cases are replaced by a cryo-EM reference. All other necessary changes to settings in Buccaneer are implemented in the model-building pipeline from within the CCP-EM interface (as of version 1.4.0).

Keywords: Buccaneer; CCP-EM; Collaborative Computational Project for Electron cryo-Microscopy; cryo-EM; model building.

Figure 1

Overview of the automated model-building pipeline using Buccaneer in the CCP-EM software suite.

Figure 2

Comparison of percentages of (a) C^α atoms built (filled circles) and (b) residues sequenced (plus symbols) correctly in one cycle of Buccaneer applying various combinations of correlation and fast modes using EM reference data. Markers above and below the diagonal dashed line correspond to better results with the respective modes turned on and off, respectively. Top: results with fast mode on versus both modes on. Middle: results with correlation mode on versus both modes on. Bottom: results with both modes off versus both modes on.

Figure 3

Comparison of percentages of (a) C^α atoms built (filled circles) and (b) residues sequenced (plus symbols) by Buccaneer using X-ray versus EM reference data. Markers above and below the diagonal dashed line correspond to better results from using X-ray and EM reference data, respectively.

Figure 4

Percentage of (a) C^α atoms built and (b) residues sequenced by Buccaneer across resolutions of 1.8–3.97 Å. Lines with markers show the means for the resolution bins 1.8–2.8 Å and 2.8–3.0 Å and for resolution bins of 0.1 Å in size from 3.0 to 4.0 Å. Areas filled with colour indicate the standard deviation of values within each bin.

Figure 5

Average success rates in sequencing fragments of different lengths for 109 models using two different chain traces and reference structures. (a) Fragments cut from the deposited models sequenced using an EM reference structure. (b) Fragments cut from the deposited models sequenced using an X-ray reference structure. (c) Fragments from the Buccaneer models sequenced using an EM reference structure. (d) Fragments from the Buccaneer models sequenced using an X-ray reference structure.

Figure 6

Estimated local resolution for the map from EMD-8912 calculated using ResMap. The surface view was generated using UCSF Chimera (Pettersen et al., 2004 ▸) with a 0.06 contour level.

Figure 7

Comparing parts of the deposited model (PDB entry 6du8) and the autobuilt model built into a map from EMD-8912. (a) Overall view of the deposited model. Close-up views are shown of (b) a β-strand region and (c) a helix part from the map. The contour level used is 0.14.

Figure 8

Comparing the deposited model (PDB entry 6evz) and the autobuilt model built into a map from EMD-3964. (a) Overall view of the deposited model. Close-up views are shown of (b) a helix chain and (c) a β-strand region from the map. The contour level used is 0.16. Circled regions show extra residues built incorrectly into the chain. The red X in the sequence indicates the location of an extra built residue.

Figure 9

Comparing the deposited model (PDB entry 5t4d) and the autobuilt model built into a map from EMD-8354. (a) Overall view of the deposited model. Close-up views are shown of (B) a helix chain and (c) a β-strand region from the map. The contour level used is 0.05.

Figure 10

A view of the amount of fragments built in a noisy map. Autobuilt models are shown for EMD-8912 built on (a) an unmasked map and (b) a masked map. Models are represented as C^α backbone strands. The map surface view was generated with a 0.06 contour level.

Figure 11

An example of the Buccaneer results tab from the CCP-EM GUI. Tabulated statistics reported by Buccaneer are shown with a graph showing the estimated model completeness with average FSC values for each pipeline cycle.