Here, a macromolecule-centred approach to three-dimensional structure determination as implemented in REFMAC5 is considered. The use of restraints to transfer chemical and structural information during macromolecular refinement, and how different sources of information can be combined in order to achieve models that are more consistent with data derived from a variety of experimental techniques, including macromolecular crystallography, cryo-EM and NMR spectroscopy, are discussed.

REFMAC5 and related tools for the refinement of atomic models into cryo-EM reconstructions in CCP-EM are reviewed. An upper bound on the correlation between observed and calculated Fourier coefficients is identified, and the practical utility of map blurring/sharpening is discussed. The Divide and Conquer pipeline for refining large complexes in parallel, and ProSHADE for the identification of symmetries in a given map or coordinate set, are presented.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.The DNA-packaging motor in tailed bacteriophages requires nuclease activity to ensure that the genome is packaged correctly. This nuclease activity is tightly regulated as the enzyme is inactive for the duration of DNA translocation. Here, we report the X-ray structure of the large terminase nuclease domain from bacteriophage SPP1. Similarity with the RNase H family endonucleases allowed interactions with the DNA to be predicted. A structurebased alignment with the distantly related T4 gp17 terminase shows the conservation of an extended b-sheet and an auxiliary b-hairpin that are not found in other RNase H family proteins. The model with DNA suggests that the b-hairpin partly blocks the active site, and in vivo activity assays show that the nuclease domain is not functional in the absence of the ATPase domain. Here, we propose that the nuclease activity is regulated by movement of the b-hairpin, altering active site access and the orientation of catalytically essential residues.

Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. Here we report the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave's propagation within the AAA+ domains is not necessarily coupled with a strictly sequential hydrolysis of ATP. Since a single ATP hydrolysis event would affect the whole hexamer, such events may simply serve to rectify the direction of the wave's motion.