Current developments in Coot for macromolecular model building of Electron Cryo-microscopy and Crystallographic Data

Abstract

Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo-EM model building and refinement, as cryo-EM structures with resolution ranging 2.5-4 A are now routinely available. Model building into these maps can be time-consuming and requires experience in both biochemistry and building into low-resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman-McClure restraints, full-chain refinement, and Fourier-model based residue-type-specific Ramachandran restraints. Here, we present the current state-of-the-art in Coot usage.

Keywords: ligands; macromolecular model building; molecular biophysics; real space refinement; rotamers; validation.

Figure 1

Steps to fit full domains or chains using Coot. After blurring the map (200 A²) and placing a homologue near the domain of interest using “move molecule here” (a), jiggle fit (b) improves the fit into the map (c). Geman‐McClure restrains (limit 6 Å) are then applied (d) to perform chain refine (e) to obtain a final accurate fit (f). Map used for representation: EMD‐3908.44 Fitted homologue: PDB 6f9n45

Figure 2

The backrub method. (a) Schematic representation of the backrub motion. The central residue and adjacent peptides move around backrub vector (yellow circle and axes). Individual adjacent peptides back‐rotate around the peptide vectors (pink). This motion preserves the geometry of the main chain while fitting the side chains into the map correctly. The algorithm in action in Coot is illustrated in panels (b), (c), and (d). In (c), the backrub option is inactive and the main chain is heavily distorted (appearance of red flags indicate that a cis‐peptide has been introduced). When the backrub option is active in Coot, the side chain is suitably fitted (d). Map and model used for representation: EMD‐3908, PDB 6eoj44

Figure 3

Ramachandran plots in Coot. (a) and (b) Ramachandran window in Coot. (a) Ramachandran plot. Each residue of the selected molecule is plotted on the canvas with the preferred (salmon), allowed (beige), and outlier (grey) regions plotted in the background. A selection box below the plot allows display of a selection of residues only. The “Outlier Only” buttons toggle to display all residues or outliers only. Below the plot, the statistics are shown. A menu bar allows, for example, printing of the plot, changing to Kleywegt plot (see b). (b) Kleywegt plot.42 Distances between NCS‐related residues are shown in the Ramachandran plot as in (a). The chains to be compared can be selected and the plot updates accordingly. (c) The different Ramachandran plots currently used for backbone validation in Coot: all, general, isoleucine/valine (Ile/Val), glycine (Gly), pre‐proline (pre‐Pro), and Proline (Pro). Colors represent same features as described in (a) and (b)