REFMAC5 for the refinement of macromolecular crystal structures

Abstract

This paper describes various components of the macromolecular crystallographic refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS experimental diffraction data. To ensure chemical and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolutions at least as low as 4 Å can be achieved thanks to low-resolution refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, `jelly-body' restraints and the use of novel long-range restraints on atomic displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 additionally offers TLS parameterization and, when high-resolution data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resolution spectrum encountered in macromolecular crystallography.

Figure 1

Fraction of the model correctly built by ARP/wARP v.7.0 iterated with REFMAC5 using different target functions. The maps inputted to model building were prepared by CRANK (Ness et al., 2004 ▶). The sample consists of 102 data sets described in Skubák et al. (2010 ▶).

Figure 2

Behaviour of the Geman–McClure function versus the quadratic (least-squares) function. For small values of r they look similar, whereas for large values of r GM is less restrictive than least squares, allowing conformational changes to occur. Black line, GM = r ²/(1 + σ² r ²) with σ = 0.5; red line, quadratic function r ². This figure was produced using the software package R (R Development Core Team, 2007 ▶).

Figure 3

Superposition of the structures of bluetongue virus VP4 enzyme with PDB entries 2jha (green) and 2jhp (blue) (Sutton et al., 2007 ▶), which were solved at 3.4 and 2.5 Å, respectively. The graph shows the resultant R (solid) and R _free (dashed) values from ten iterations of refinement of the low-resolution structure 2jha. Results are shown with (red) and without (black) external restraints, using 2jhp as prior information. This figure was produced using PROSMART to superpose the structures, PyMOL (DeLano, 2002 ▶) to display the structures and the software package R (R Development Core Team, 2007 ▶) to generate the graph.

Figure 4

TLS refinement of glucosamine-6-phosphate synthase (Mouilleron & Golinelli-Pimpaneau, 2007 ▶). The results for chain C are shown, which is separated into two TLS groups. Thermal ellipsoids derived from the TLS refinement are shown for the two groups. Those in red correspond to the ligand Fru6P which is included in the TLS group for the synthase domain. The yellow arrows show the principal axes of the libration tensor for each TLS group. Inclusion of TLS parameters led to a reduction in R and R _free of 3.4% and 3.8%, respectively, and could be related to the biological function. The principal axis of the libration tensor was calculated using TLSANL (Howlin et al., 1993 ▶) and the figure was prepared using CCP4mg (Potterton et al., 2004 ▶).