A key step in proliferation of retroviruses is the conversion of their RNA genome to double-stranded DNA, a process catalysed by multifunctional reverse transcriptases (RTs). Dimeric and monomeric RTs have been described, the latter exemplified by the enzyme of Moloney murine leukaemia virus. However, structural information is lacking that describes the substrate binding mechanism for a monomeric RT. We report here the first crystal structure of a complex between an RNA/DNA hybrid substrate and polymerase-connection fragment of the single-subunit RT from xenotropic murine leukaemia virus-related virus, a close relative of Moloney murine leukaemia virus. A comparison with p66/p51 human immunodeficiency virus-1 RT shows that substrate binding around the polymerase active site is conserved but differs in the thumb and connection subdomains. Small-angle X-ray scattering was used to model full-length xenotropic murine leukaemia virus-related virus RT, demonstrating that its mobile RNase H domain becomes ordered in the presence of a substrate—a key difference between monomeric and dimeric RTs.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.

A nonredundant set of 9081 protein crystal structures in the Protein Data Bank was used to examine the solvent content, the number of polypeptide chains, and the oligomeric states of proteins in crystals as a function of crystal symmetry (as classified by crystal systems and space groups). It was found that there is a correlation between solvent content and crystal symmetry. Surprisingly, proteins crystallizing in lower symmetry systems have lower solvent content compared to those crystallizing in higher symmetry systems. Nevertheless, there is no universal correlation between solvent content and preferences of macromolecules to crystallize in certain space groups. Crystal symmetry as a function of oligomeric state was examined, where trimers, tetramers, and hexamers were found to prefer to crystallize in systems where the oligomer symmetry could be incorporated in the crystal symmetry. Our analysis also shows that the frequency distribution within the enantiomorphous pairs of space groups does not differ significantly, in contrast to previous reports.Keywords: solvent content; Matthews coefficient; protein crystals; oligomerization; space group frequency Supplemental material: see www.proteinscience.orgWater plays an important role in the structure of biomolecules and often influences protein function. Water molecules not only affect protein folding, but also mediate biological processes such as enzymatic reactions and molecular recognition. Information about the fraction of water (solvent) plays a significant role in the X-ray structure determination process. First, knowledge of the solvent content helps to determine the number of molecules in the asymmetric unit (Matthews 1968), which is crucial in early stages of crystal structure determination. Second, an approximate value of solvent content is needed for significant phase improvement by solvent flattening methods (Wang 1985;Leslie 1987;Abrahams and Leslie 1996), which is necessary to resolve the inherent phase ambiguity in single anomalous diffraction (SAD) experiments. For both SAD and MAD (multiwavelength anomalous diffraction) (Hendrickson 1991;Hendrickson et al. 1990), phase improvement by solvent flattening is critical for low resolution data (Kirillova et al. 2007), especially when non-crystallographic symmetry cannot be applied.Matthews (1968) observed that the solvent content in protein crystals ranged from 27% to 65%, with an average of 43%. He also showed that the quantity V M (the Matthews coefficient, defined as the ratio of the volume of the asymmetric unit to the molecular weight of all