Developments in low-resolution biological X-ray crystallography

Despite the recent substantial technological developments in X-ray crystallography, solving and refining structures at low resolutions remain substantial challenges. Many macromolecular crystals, especially those of large molecules or multicomponent assemblies, diffract X-rays to resolutions that are worse than 3.5Å. This report summarizes several recent advances aiding low resolution crystallographic work

CRISPR-Cas immunity and mobile DNA: a new superfamily of DNA transposons encoding a Cas1 endonuclease

Mobile genetic elements such as DNA transposons are a feature of most genomes. The existence of novel DNA transposons can be inferred when whole genome sequencing reveals the presence of hallmarks of mobile elements such as terminal inverted repeats (TIRs) flanked by target site duplications (TSDs). A recent report describes a new superfamily of DNA transposons in the genomes of a few bacteria and archaea that possess TIRs and TSDs, and encode several conserved genes including a cas1 endonuclease gene, previously associated only with CRISPR-Cas adaptive immune systems. The data strongly suggests that these elements, designated ‘casposons’, are likely to be bona fide DNA transposons and that their Cas1 nucleases act as transposases and are possibly still active

Molecular Organization in Site-Specific Recombination: The Catalytic Domain of Bacteriophage HP1 Integrase at 2.7 Å Resolution

AbstractHP1 integrase promotes site-specific recombination of the HP1 genome into that of Haemophilus influenzae. The isolated C-terminal domain (residues 165–337) of the protein interacts with the recombination site and contains the four catalytic residues conserved in the integrase family. This domain represents a novel fold consisting principally of well-packed α helices, a surface β sheet, and an ordered 17-residue C-terminal tail. The conserved triad of basic residues and the active-site tyrosine are contributed by a single monomer and occupy fixed positions in a defined active-site cleft. Dimers are formed by mutual interactions of the tail of one monomer with an adjacent monomer; this orients active-site clefts antiparallel to each other

An Improved Model for Dynamin Assembly Revealed by Cryo-EM

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Water molecules in the antibody朼ntigen interface of the structure of the Fab HyHEL-5杔ysozyme complex at 1.7 A? resolution: comparison with results from isothermal titration calorimetry

The structure of the complex between hen egg-white lysozyme and the Fab HyHEL-5 at 2.7 A? resolution has previously been reported [Cohen et al. (1996), Acta Cryst. D52, 315�6]. With the availability of recombinant Fab, the X-ray structure of the complex has been re-evaluated at 1.7 A? resolution. The refined structure has yielded a detailed picture of the Fab� lysozyme interface, showing the high complementarity of the protein surfaces as well as several water molecules within the interface that complete the good fit. The model of the full complex has improved significantly, yielding an Rwork of 19.5%. With this model, the structural results can be compared with the results of isothermal titration calorimetry. An attempt has been made to estimate the changes in bound waters that accompany complex formation and the difficulties inherent in using the crystal structures to provide the information necessary to make this calculation are discussed

Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5′ to the foot of each IP, guide (G) sequences, using a network of canonical (‘Watson–Crick’) base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions

Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding

Abstract The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end

The Structural Basis of Ordered Substrate Binding by Serotonin N-Acetyltransferase Enzyme Complex at 1.8 Å Resolution with a Bisubstrate Analog

AbstractSerotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance