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. 2018 Mar 1;74(Pt 3):215-227.
doi: 10.1107/S2059798318000979. Epub 2018 Mar 2.

Overview of refinement procedures within REFMAC5: utilizing data from different sources

Oleg Kovalevskiy  1 , Robert A Nicholls  1 , Fei Long  1 , Azzurra Carlon  2 , Garib N Murshudov  1
Affiliations

Overview of refinement procedures within REFMAC5: utilizing data from different sources

Oleg Kovalevskiy et al. Acta Crystallogr D Struct Biol. .

Abstract

Refinement is a process that involves bringing into agreement the structural model, available prior knowledge and experimental data. To achieve this, the refinement procedure optimizes a posterior conditional probability distribution of model parameters, including atomic coordinates, atomic displacement parameters (B factors), scale factors, parameters of the solvent model and twin fractions in the case of twinned crystals, given observed data such as observed amplitudes or intensities of structure factors. A library of chemical restraints is typically used to ensure consistency between the model and the prior knowledge of stereochemistry. If the observation-to-parameter ratio is small, for example when diffraction data only extend to low resolution, the Bayesian framework implemented in REFMAC5 uses external restraints to inject additional information extracted from structures of homologous proteins, prior knowledge about secondary-structure formation and even data obtained using different experimental methods, for example NMR. The refinement procedure also generates the `best' weighted electron-density maps, which are useful for further model (re)building. Here, the refinement of macromolecular structures using REFMAC5 and related tools distributed as part of the CCP4 suite is discussed.

Keywords: LORESTR; NMR restraints; ProSMART; REFMAC5; refinement.

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Figures

Figure 1
Figure 1
Experimental data and complementary prior knowledge versus resolution. The grey arrow shows resolution in Å and the grey bar above it indicates the level of macromolecular structural detail that can be observed in the corresponding resolution range. The blue panel shows experimental methods that deliver information in the corresponding resolution range; the NMR bar is coloured with a pattern to reflect the different nature of NMR-derived data (distances between points rather than a continuous distribution of density). The green panel shows structural restraints that are useful for complementing the experimental data that are missing at certain resolutions. The top arrow indicates the minimal theoretical resolvability of peaks in the absence of noise at a given resolution.
Figure 2
Figure 2
Automated refinement of PDB entry 1jkt with the LORESTR pipeline and comparative structural analysis using ProSMART. The figure shows superposition of the model with PDB entry 1jkt before and after automated re-refinement (the corresponding refinement statistics are shown in Table 1 ▸). Both structures are coloured according to local backbone deviation using ProSMART (as described by Nicholls et al., 2014 ▸). This representation allows quick and easy visual identification of exactly which regions have changed during re-refinement. The colour gradient indicates local backbone r.m.s. deviation in the vicinity of each residue; residues coloured yellow retain their conformation, whilst those coloured red exhibit substantial structural changes during refinement. Two regions that have undergone dramatic local structural changes are highlighted by grey ovals. In practice, the electron-density maps in such regions would be manually inspected in order to assess the reasons for such changes to the model and to determine how best to proceed with further model building and refinement. The figure was prepared using CCP4mg (McNicholas et al., 2011 ▸).
Figure 3
Figure 3
NMR-based structural restraints commonly used in model refinement. (a) NOEs provide short-range interatomic distances, (b) PCSs give long-range distance and angular information of the metal–nucleus vectors relative to an external reference frame, and (c) RDCs provide angular information corresponding to vectors connecting two nuclei relative to the external reference frame. In cases where the alignment of the molecule arises from the presence of a paramagnetic centre, PCS and RDC restraints have the same reference frame.
Figure 4
Figure 4
Refinement of the ternary Sxl–Unr–msl2-mRNA regulatory complex. (a) The structure refined using the REFMAC5 protocol is coloured according to the difference (in absolute value) between experimental RDCs and those back-calculated from the model. No RDCs were measured for residues coloured in white. (b, c, d) Enlarged views for residues reporting the largest differences. Reprinted (adapted) with permission from Carlon et al. (2016 ▸). Copyright (2016) American Chemical Society.
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