Real-space refinement in PHENIX for cryo-EM and crystallography

Abstract

This article describes the implementation of real-space refinement in the phenix.real_space_refine program from the PHENIX suite. The use of a simplified refinement target function enables very fast calculation, which in turn makes it possible to identify optimal data-restraint weights as part of routine refinements with little runtime cost. Refinement of atomic models against low-resolution data benefits from the inclusion of as much additional information as is available. In addition to standard restraints on covalent geometry, phenix.real_space_refine makes use of extra information such as secondary-structure and rotamer-specific restraints, as well as restraints or constraints on internal molecular symmetry. The re-refinement of 385 cryo-EM-derived models available in the Protein Data Bank at resolutions of 6 Å or better shows significant improvement of the models and of the fit of these models to the target maps.

Keywords: PHENIX; atomic-centered targets; cryo-EM; crystallography; map interpolation; real-space refinement.

Figure 1

Number of cryo-EM-derived models in the PDB at resolutions of 6 Å or better.

Figure 2

Flowchart for phenix.real_space_refine.

Figure 3

Refinement of the exact model against 18 maps computed as described in §3.1.1. Each circle shows the root-mean-square deviation between the refined model and the reference model. Blue, green and orange full circles correspond to maps with overall B factors of 0, 100 and 200 Ų, respectively. Open circles correspond to the map with an overall B factor of 100 Ų computed on the finer grid with a step of 0.2 Å. See §3.1.2 for details.

Figure 4

Refinement of perturbed models against maps computed as described in §3.1.1. The horizontal axis shows the r.m.s.d. between the reference model and perturbed models: 0.5, 1.0, 1.5 and 2.0 Å. The vertical axis shows the r.m.s.d. between the reference model and the refined models. Blue, green and orange full circles correspond to maps with overall B factors of 0, 100 and 200 Ų, respectively. See §3.1.3 for details.

Figure 5

Distribution of cryo-EM map values (scaled in r.m.s.) for selected groups of atoms, considering maps at 3 Å or better (a) and 3–4 Å (b) resolution. See §3.2.1 for details.

Figure 6

Model statistics before (brown) and after (blue) refinement using phenix.real_space_refine, showing Ramachandran plot and residue side-chain rotamer outliers, C^β deviations, MolProbity clashscore and model–map correlation coefficient (CC_mask). The scatter plot shows the EMRinger score for the original and refined models (resolution better than 4.5 Å).

Figure 7

Left, correlation coefficient CC_mask calculated using the original second half-maps and maps calculated from models refined against the first half-maps: original (x axis) versus sharpened (y axis). Right, MolProbity scores for models using original first half-maps versus sharpened first half-maps.

Figure 8

Backbone of the 3j5p model before (a) and after (b) refinement shown in black. The model before refinement contains a substantial number of steric clashes (indicated by red dots) and many side-chain rotamer outliers (blue side chains). Most clashes and rotamer outliers are resolved by phenix.real_space_refine. The images were created using the KiNG program (Chen et al., 2009 ▸) from within PHENIX.

Figure 9

(a) Ensemble of perturbed 3j5p models; the r.m.s. deviation of each model from the initial model is 3 Å, showing chain A only. (b) Ensemble of refined models in the experimental map. The largest variation is observed in regions that lack density. The images were created using the ChimeraX program (Goddard et al., 2018 ▸).