Accelerated X-ray Structure Elucidation of a 36 kDa Muramidase/Transglycosylase Using wARP

The X-ray structure of the 36kDa soluble lytic transglycosylase from Escherichia coli has been determined starting with the multiple isomorphous replacement method with inclusion of anomalous scattering at 2.7 Å resolution. Subsequently, before any model building was carried out, phases were extended to 1.7 Å, resolution with the weighted automated refinement procedure wARP, which gave a dramatic improvement in the phases. The electron-density maps from wARP were of outstanding quality for both the main chain and the side chains of the protein, which allowed the time spent on the tracing, interpretation and building of the X-ray structure to be substantially shortened. The structure of the soluble lyric transglycosylase was refined at 1.7 Å, resolution with X-PLOR to a final crystallographic R factor of 18.9%. Analysis of the wARP procedure revealed that the use of the maximum-likelihood refinement in wARP gave much better phases than least-squares refinement, provided that the ratio of reflections to protein atom parameters was approximately 1.8 or higher. Furthermore, setting aside 5% of the data for an Rfree test set had a negative effect on the phase improvement. The mean WwARP, a weight determined at the end of the wARP procedure and based on the variance of structure factors from six individually refined wARP models, proved to be a better indicator than the Rfree factor to judge different phase improvement protocols. The elongated Slt35 structure has three domains named the alpha, beta and core domains. The alpha domain contains mainly α-helices, while the beta domain consists of a five-stranded antiparallel β-sheet flanked by a short α-helix. Sandwiched between the alpha and beta domains is the core domain, which bears some resemblance to the fold of the catalytic domain of the previously elucidated 70 kDa soluble lytic transglycosylase from E. coli. The putative active site is at the bottom of a large deep groove in the core domain.

Pioneering Robotic Liver Surgery in Germany: First Experiences with Liver Malignancies

Background Minimally invasive liver surgery is growing worldwide with obvious benefits for the treated patients. These procedures maybe improved by robotic techniques, which add several innovative features. In Germany, we were the first surgical department implementing robotic assisted minimally invasive liver resections. Material and methods Between June 2013 and March 2015, we performed robotic based minimally invasive liver resections in nine patients with malignant liver disease. Five off these patients suffered from primary and four from secondary liver malignancies. We retrospectively analyzed the perioperative variables of these patients and the oncological follow up. Results Mean age of the patients was 63 years (range 45–71). One patient suffered from intrahepatic cholangiocellular, four from hepatocellular carcinoma, and four patients from colorectal liver metastases. In six patients, left lateral liver resection, in two cases single segment resection, and in one case minimally invasive guided liver ablation were performed. Five patients underwent previous abdominal surgery. Mean operation time was 312 min (range 115–458 min). Mean weight of the liver specimens was 182 g (range 62–260 g) and mean estimated blood loss was 251 ml (range 10–650 ml). The mean tumor size was 4.4 cm (range 3.5–5.5 cm). In all cases, R0 status was confirmed with a mean margin of 0.6 cm (range 0.1–1.5 cm). One patient developed small bowel fistula on postoperative day 5, which could be treated conservatively. No patient died. Mean hospital stay of the patients was 6 days (range 3–10 days). During a mean follow up of 12 months (range 1–21 months), two patients developed tumor recurrence. Conclusion Robotic-based liver surgery is feasible in patients with primary and secondary liver malignancies. To achieve perioperative parameters comparable to open settings, the learning curve must be passed. Minor liver resections are good candidates to start this technique. But the huge benefits of robotic-based liver resections should be expected in extended procedures beyond minor liver resections with the currently available technology

Introductory report [on European citizenship, fundamental rights and the third pillar].

This report introduced the proceedings of a symposium session that was devoted to an examination of the subjects of European Union citizenship, fundamental rights and Title VI (third pillar) Treaty on the European Union (TEU) in the context of the 1996 Intergovernmental Conference. There are strong links between these three important issues and other sessions of the symposium, in particular 'Elaboration of a fundamental treaty' (Session 1) and 'Democratization of the European Union' (Session 2). However, these three subjects were considered to be deserving of independent treatment given their widely perceived importance for the future of the Union. The report is followed by notes from the discussion, a summary of conclusions drawn during the session, and a list of other participants and their papers

Substrate-Assisted Catalysis Unifies Two Families of Chitinolytic Enzymes

Hen egg-white lysozyme has long been the paradigm for enzymatic glycosyl hydrolysis with retention of configuration, with a protonated carboxylic acid and a deprotonated carboxylate participating in general acid-base catalysis. In marked contrast, the retaining chitin degrading enzymes from glycosyl hydrolase families 18 and 20 all have a single glutamic acid as the catalytic acid but lack a nucleophile on the enzyme. Both families have a catalytic (βα)8-barrel domain in common. X-ray structures of three different chitinolytic enzymes complexed with substrates or inhibitors identify a retaining mechanism involving a protein acid and the carbonyl oxygen atom of the substrate’s C2 N-acetyl group as the nucleophile. These studies unambiguously demonstrate the distortion of the sugar ring toward a sofa conformation, long postulated as being close to that of the transition state in glycosyl hydrolysis.

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein

Campylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain Barre syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two-domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a K-d of similar to 10(-7) M consistent with a role as a receptor for a high- affinity transporter. These data imply that Cj0982 is the binding protein component of an ABC-type cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni

Analogy making and the structure of implied volatility skew

An analogy based option pricing model is put forward. If option prices are determined in accordance with the analogy model, and the Black Scholes model is used to back-out implied volatility, then the implied volatility skew arises, which flattens as time to expiry increases. The analogy based stochastic volatility and the analogy based jump diffusion models are also put forward. The analogy based stochastic volatility model generates the skew even when there is no correlation between the stock price and volatility processes, whereas, the analogy based jump diffusion model does not require asymmetric jumps for generating the skew

Structure and mechanism of acetolactate decarboxylase

Acetolactate decarboxylase catalyzes the conversion of both enantiomers of acetolactate to the (R)-enantiomer of acetoin, via a mechanism that has been shown to involve a prior rearrangement of the non-natural (R)-enantiomer substrate to the natural (S)-enantiomer. In this paper, a series of crystal structures of ALDC complex with designed transition state mimics are reported. These structures, coupled with inhibition studies and site-directed mutagenesis provide an improved understanding of the molecular processes involved in the stereoselective decarboxylation/protonation events. A mechanism for the transformation of each enantiomer of acetolactate is proposed

wARP

wARP is a procedure that substantially improves crystallographic phases (and subsequently electron-density maps) as an additional step after density-modification methods such as solvent flattening and averaging. The initial phase set is used to create a number of dummy atom models which are subjected to least-squares or maximum-likelihood refinement and iterative model updating in an automated refinement procedure (ARP). Averaging of the phase sets calculated from the refined output models and weighting of structure factors by their similarity to an average vector results in a phase set that improves and extends the initial phases substantially. An important requirement is that the native data have a maximum resolution beyond \sim2.4 Å. The wARP procedure shortens the time-consuming step of model building in crystallographic structure determination and helps to prevent the introduction of errors

Comparative structural and functional analysis of Bunyavirus and Arenavirus cap-snatching Endonucleases

Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5′ end by an endonuclease (EN) domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively), but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase