Low-resolution refinement tools in REFMAC5

Abstract

Two aspects of low-resolution macromolecular crystal structure analysis are considered: (i) the use of reference structures and structural units for provision of structural prior information and (ii) map sharpening in the presence of noise and the effects of Fourier series termination. The generation of interatomic distance restraints by ProSMART and their subsequent application in REFMAC5 is described. It is shown that the use of such external structural information can enhance the reliability of derived atomic models and stabilize refinement. The problem of map sharpening is considered as an inverse deblurring problem and is solved using Tikhonov regularizers. It is demonstrated that this type of map sharpening can automatically produce a map with more structural features whilst maintaining connectivity. Tests show that both of these directions are promising, although more work needs to be performed in order to further exploit structural information and to address the problem of reliable electron-density calculation.

Tackling the crystallographic structure determination of the COP9 signalosome

“…The co-refinement approach used here was inspired by an implementation in MAIN (Turk, 2013). Proposed methods extending this to simultaneous cross-crystal refinement with electron-density averaging (Brunger, 2005;Nicholls et al, 2012) Representative electron density for CSN. A portion of the electrondensity map (grey mesh) surrounding the active site of CSN5 (cyan) calculated at 4 Å resolution and contoured at 1 r.m.s.…”

“…The untwinned structures, c318 and c337, were refined using autoBUSTER with reference-model restraints applied as LSSR. The twinned structure, c343, was refined by REFMAC (Murshudov et al, 2011) with reference-model restraints generated by PROSMART (Nicholls et al, 2012). Co-refinement was carried out using reference-model restraints generated across crystals, typically between crystals c318 and c337 and between crystals c337 and c343, to parts of the model lacking restraints to high-resolution CSN4 and CSN5 models.…”

Recent developments in theCCP-EMsoftware suite

“…Initially developed for lowresolution crystallography, additional structural restraints can be helpful when the information content in the map density is low. ProSMART (Nicholls et al, 2012) can used to generate such additional restraints for REFMAC. Flex-EM requires rigid-body definitions and these can be produced using the helper program RIBFIND (Pandurangan & Topf, 2012a,b).…”

“…ProSMART (Procrustes Structural Matching Alignment and Restraint Tool) was originally developed by Nicholls et al (2012) to aid the modelling of low-resolution X-ray structures via the generation of additional restraints, and has since been successfully applied to model refinement from cryo-EM data (Brown et al, 2015). The external restraints generated by ProSMART can be added to REFMAC to supplement its standard dictionary of restraints.…”

“…This task runs the 'sfcalc' mode of REFMAC and has two specific roles: the conversion of real-space maps in MRC format to reciprocal-space structure factors in MTZ format, and map sharpening/blurring (Nicholls et al, 2012). Presently, the optimum sharpening coefficient (where 'optimum' means maximizing the interpretable density features) cannot be analytically determined either locally or globally, although attempts are ongoing.…”