Alternate Structural Conformations of Streptococcus pneumoniae Hyaluronan Lyase: Insights into Enzyme Flexibility and Underlying Molecular Mechanism of Action.

Streptococcus pneumoniae hyaluronan lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades several biologically important, information-rich linear polymeric glycans: hyaluronan, unsulfated chondroitin, and some chondroitin sulfates. This degradation facilitates spreading of bacteria throughout the host tissues and presumably provides energy and a carbon source for pneumococcal cells. Its β-elimination catalytic mechanism is an acid/base process termed proton acceptance and donation leading to cleavage of β-1,4 linkages of the substrates. The degradation of hyaluronan occurs in two stages, initial endolytic cuts are followed by processive exolytic cleavage of one disaccharide at a time. In contrast, the degradation of chondroitins is purely endolytic. Structural studies together with flexibility analyses of two streptococcal enzymes, from S. pneumoniae and Streptococcus agalactiae, allowed for insights into this enzyme's molecular mechanism. Here, two new X-ray crystal structures of the pneumococcal enzyme in novel conformations are reported. These new conformations, complemented by molecular dynamics simulation results, directly confirm the predicted domain motions presumed to facilitate the processive degradative process. One of these new structures resembles the S. agalactiae enzyme conformation, and provides evidence of a uniform mechanistic/dynamic behavior of this protein across different bacteria

Monomorphic subtelomeric DNA in the filamentous fungus, Metarhizium anisopliae, contains a RecQ helicase-like gene.

In most filamentous fungi, telomere-associated sequences (TASs) are polymorphic, and the presence of restriction fragment length polymorphisms (RFLPs) may permit the number of chromosome ends to be estimated from the number of telomeric bands obtained by restriction digestion. Here, we describe strains of Metarhizium, Gliocladium and Paecilomyces species in which only one or a few telomeric bands of unequal intensity are detectable by Southern hybridization, indicating that interchromosomal TAS exchange occurs. We also studied an anomalous strain of Metarhizium anisopliae, which produces polymorphic telomeric bands larger than 8 kb upon digestion of genomic DNA with XhoI. In this case, the first XhoI site in from the chromosome end must lie beyond the presumed monomorphic region. Cloned telomeres from this strain comprise 18?26 TTAGGG repeats, followed at the internal end of the telomere tract by five repeats of the telomere-like sequence TAAACGCTGG. An 8.1-kb TAS clone also contains a gene for a RecQ-like helicase, designated TAH1, suggesting that this TAS is analogous to the Y elements in yeast and the subtelomeric helicase ORFs of Ustilago maydis (UTASRecQ) and Magnaporthe grisea (TLH1). The TAS in the anomalous strain of M. anisopliae, however, appears distinct from these in that it is found at most telomeres and its predicted protein product possesses a significantly longer N-terminal region in comparison to the M. grisea and U. maydis helicases. Hybridization analyses showed that TAH1 homologues are present in all other anomalous M. anisopliae strains studied, as well as in some other polymorphic strains, where the recQ-like gene also appears to be telomere-associated.Published online: 2 June 2005

Mildred Dresselhaus and Solid State Pedagogy at MIT

Mildred Dresselhaus is known for her influential research on the physics of carbon. Her wide‐ranging influence as a physics teacher, although well‐known to her students, has been less thoroughly examined. Exploring how Dresselhaus grew into her role teaching solid state physics at MIT reveals much about how that subfield evolved

Exploring the speed and performance of molecular replacement with AMPLE using QUARK ab initio protein models

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110744/1/S1399004714025784.pd

Comparison between methods for creating DEMs of physical models

Within physical modelling, it is often necessary to create DEMs (digital elevation models) when testing the stability of rock structures or the filter layers and scour protection around foundations and other marine structures. These DEMs are used to detect changes in the position of the structure or surrounding protective material. Several methods are available to create these models, yet no one technique has been selected as an industry standard. A comparison between three widely used methods – terrestrial laser scanner (TLS), combined laser scanner (CLS) and structure from motion (SfM) – are presented within this paper. The CLS in underwater mode gave low measurement errors and can be deployed without having to drain the facility but requires a traverser system. An area of approximately 7 m by 4 m can be measured in half an hour. The TLS can survey a much larger area in the same time, but requires the facility to be drained. SfM is cheapest method, but struggles to create a full shape and more care must be taken. The CLS in underwater mode has been chosen for use in scour studies in the Fast Flow Facility, with high volumes of water but a relatively limited area

Routine phasing of coiled-coil protein crystal structures with AMPLE

Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallographic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing

Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests.

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Assessing the utility of CASP14 models for molecular replacement

Funder: CCP4Funder: Max‐Planck‐Gesellschaft; Id: http://dx.doi.org/10.13039/501100004189Abstract: The assessment of CASP models for utility in molecular replacement is a measure of their use in a valuable real‐world application. In CASP7, the metric for molecular replacement assessment involved full likelihood‐based molecular replacement searches; however, this restricted the assessable targets to crystal structures with only one copy of the target in the asymmetric unit, and to those where the search found the correct pose. In CASP10, full molecular replacement searches were replaced by likelihood‐based rigid‐body refinement of models superimposed on the target using the LGA algorithm, with the metric being the refined log‐likelihood‐gain (LLG) score. This enabled multi‐copy targets and very poor models to be evaluated, but a significant further issue remained: the requirement of diffraction data for assessment. We introduce here the relative‐expected‐LLG (reLLG), which is independent of diffraction data. This reLLG is also independent of any crystal form, and can be calculated regardless of the source of the target, be it X‐ray, NMR or cryo‐EM. We calibrate the reLLG against the LLG for targets in CASP14, showing that it is a robust measure of both model and group ranking. Like the LLG, the reLLG shows that accurate coordinate error estimates add substantial value to predicted models. We find that refinement by CASP groups can often convert an inadequate initial model into a successful MR search model. Consistent with findings from others, we show that the AlphaFold2 models are sufficiently good, and reliably so, to surpass other current model generation strategies for attempting molecular replacement phasing

Integration of phenotypic metadata and protein similarity in Archaea using a spectral bipartitioning approach

In order to simplify and meaningfully categorize large sets of protein sequence data, it is commonplace to cluster proteins based on the similarity of those sequences. However, it quickly becomes clear that the sequence flexibility allowed a given protein varies significantly among different protein families. The degree to which sequences are conserved not only differs for each protein family, but also is affected by the phylogenetic divergence of the source organisms. Clustering techniques that use similarity thresholds for protein families do not always allow for these variations and thus cannot be confidently used for applications such as automated annotation and phylogenetic profiling. In this work, we applied a spectral bipartitioning technique to all proteins from 53 archaeal genomes. Comparisons between different taxonomic levels allowed us to study the effects of phylogenetic distances on cluster structure. Likewise, by associating functional annotations and phenotypic metadata with each protein, we could compare our protein similarity clusters with both protein function and associated phenotype. Our clusters can be analyzed graphically and interactively online