ORCID Profile
0000-0003-2666-3219
Current Organisations
UNSW Sydney
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CSIRO
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Publisher: International Union of Crystallography (IUCr)
Date: 07-2020
Publisher: Springer New York
Date: 22-11-2015
DOI: 10.1007/978-1-4939-2230-7_8
Abstract: Crystals of biological macromolecules have been observed and grown for well over a century. More effort has been put into biological crystallization in the last few decades due to the importance of X-ray crystal structures, the advent of synchrotron radiation sources, improved computational speed, better software, and the availability of recombinant protein. Here we focus on two important areas of crystal growth: firstly, on techniques for stabilizing the protein s le, and secondly, on strategies and approaches for selecting the crystallization cocktails most suitable for different strategies.
Publisher: Elsevier BV
Date: 06-2020
Publisher: Wiley
Date: 16-09-2010
Publisher: International Union of Crystallography (IUCr)
Date: 28-09-2005
Publisher: International Union of Crystallography (IUCr)
Date: 31-10-2019
DOI: 10.1107/S2059798319013883
Abstract: Atrazine is an s -triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s -triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Å resolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.
Publisher: American Chemical Society (ACS)
Date: 12-12-2011
DOI: 10.1021/CG201206E
Publisher: International Union of Crystallography (IUCr)
Date: 16-11-2010
Publisher: American Chemical Society (ACS)
Date: 10-06-2013
DOI: 10.1021/CO400013V
Abstract: We present a high-throughput approach to help define experimental formulations that enhance protein stability, which is based on differential scanning fluorimetry (DSF). The method involves defining the thermal stability of a protein against a screen of 13 buffer systems, systematically s ling pH from 5.0 to 9.0 at high and low salt concentrations, using both redundancy and extensive controls to make the method robust. The screen allows rapid determination of a suitable base formulation for protein s les, and is particularly useful for difficult s les: those that are rapidly degraded or cannot be sufficiently concentrated for downstream analyses. Data obtained from three s les in this assay illustrate the vastly different values for thermal stability that can be obtained from different formulations. This approach is simple to interpret and reliable enough that it has been implemented as a service through the Collaborative Crystallisation Centre (C3).
Publisher: Springer Science and Business Media LLC
Date: 21-12-2012
Publisher: Springer International Publishing
Date: 2019
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7GC02343J
Abstract: The use of ancestral sequence reconstruction to design novel biocatalysts with improved catalytic properties for the production of polyamide precursors.
Publisher: American Chemical Society (ACS)
Date: 29-08-2017
Publisher: No publisher found
Date: 2011
DOI: 10.1016/J.YMETH.2011.04.004
Abstract: Turning commercial lab automation into a high-throughput centre requires an underlying process, and implementing checks to ensure that the process is working as it should. At the Collaborative Crystallisation Centre (C3), protein s les from local, national and international groups are set up in crystallisation screening and optimisation experiments with two thousand 96 well plates being set up each year. During its five years of operation, the C3 has implemented a series of enabling protocols - from simple 'reality checks' to determine if a screen has evaporated during storage to more sophisticated systems such as a s le labelling and tracking system. The most important - and perhaps surprising - lesson has been how much effort is required to effectively communicate between the centre and its clients, as well as between the centre's staff members. It is easy to confuse the concept of 'high throughput' in any field with the idea of setting up an experiment quickly. Although automation can be used to set up a single experiment more rapidly than can be done by hand, the distinguishing feature of a high throughput technology is the sustainability of the increased rate.
Publisher: Springer Science and Business Media LLC
Date: 04-03-2021
DOI: 10.1038/S41598-021-84551-9
Abstract: Puromycin and the Streptomyces alboniger -derived puromycin N -acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N -acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.
Publisher: SAGE Publications
Date: 04-2011
DOI: 10.3851/IMP1716
Abstract: HIV-1 integrase is a clinically validated therapeutic target for the treatment of HIV-1 infection, with one approved therapeutic currently on the market. This enzyme represents an attractive target for the development of new inhibitors to HIV-1 that are effective against the current resistance mutations. A fragment-based screening method employing surface plasmon resonance and NMR was initially used to detect interactions between integrase and fragments. The binding sites of the fragments were elucidated by crystallography and the structural information used to design and synthesize improved ligands. The location of binding of fragments to the catalytic core of integrase was found to be in a previously undescribed binding site, adjacent to the mobile loop. Enzyme assays confirmed that formation of enzyme–fragment complexes inhibits the catalytic activity of integrase and the structural data was utilized to further develop these fragments into more potent novel enzyme inhibitors. We have defined a new site in integrase as a valid region for the structure-based design of allosteric integrase inhibitors. Using a structure-based design process we have improved the activity of the initial fragments 45-fold.
Publisher: International Union of Crystallography (IUCr)
Date: 30-04-2019
Publisher: Elsevier BV
Date: 02-2013
Abstract: Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening c aign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two c aigns used different screening methods for the preliminary identification of fragment hits one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C6NR07634C
Abstract: The structural changes occurring at the nanoscale level within the lipid bilayer and driving the in-meso formation of large well-diffracting membrane protein crystals have been uniquely characterized for a model membrane protein, intimin. Importantly, the order to order transitions taking place within the bilayer and the lipidic nanostructures required for crystal growth have been shown to be general, occurring for both the cubic and the sponge mesophase crystallization pathways. For the first time, a transient fluid lamellar phase has been observed and unambiguously assigned for both crystallization pathways, present at the earliest stages of protein crystallogenesis but no longer observed once the crystals surpass the size of the average lyotropic liquid crystalline domain. The reported time-resolved structural investigation provides a significantly improved and general understanding of the nanostructural changes taking place within the mesophase during in-meso crystallization which is a fundamental advance in the enabling area of membrane protein structural biology.
Publisher: CSIRO Publishing
Date: 03-08-2021
DOI: 10.1071/AH20265
Abstract: Objective In 2018 the Australian Commission on Quality and Safety in Health Care released a new consumer driven Australian Hospital Patient Experience Question set. The objective was to explore the acceptability, adoption, appropriateness, feasibility, fidelity, penetration, resources and sustainability of implementing the AHPEQS, and to review which questions correlated with overall rating of care. Methods Thirty-six Australian private hospitals participated in the AHPEQS implementation over an 18-month period. Results Responses were analysed for 86 180 patient experience surveys. Positive patient experiences (Question 12) correlated most highly with Question 2 (My in idual needs were met correlation coefficient (CC) 0.45, P 0.001), Question 4 (I felt cared for CC 0.45, P 0.001) and Question 9 (When I was in the hospital, I felt confident in the safety of my treatment and care CC 0.44, P 0.001). Day procedure patients rated their experience higher than overnight patients. Uptake was rapid across the 36 hospitals, with minimal resources and demonstrable acceptability, adoption, appropriateness, feasibility, fidelity, penetration and sustainability. Conclusion Utilising a consumer-driven survey highlighting treatment and care, assisted in enhancing staff engagement and continuous improvement in patient experience in acute, day procedure and rehabilitation hospitals. What is known about the topic? Patient experience in hospital is associated with healthcare quality, safety and outcomes. What does this paper add? From a consumer perspective, feeling cared for, having needs met and confidence in the safety of care, correlated with a positive patient experience. What are the implications for practitioners? Investing staff time and health service resources into a consumer-driven patient experience survey tool, which highlighted treatment and care, assisted in enhancing staff engagement and continuous improvement in patient experience in acute, day procedure and rehabilitation hospitals.
Publisher: American Society for Microbiology
Date: 15-01-2015
DOI: 10.1128/AEM.02783-14
Abstract: The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s -triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis , when purified enzyme was tested in vitro . However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.
Publisher: American Chemical Society (ACS)
Date: 04-02-2013
DOI: 10.1021/CG301755A
Publisher: International Union of Crystallography (IUCr)
Date: 19-11-2005
Publisher: International Union of Crystallography (IUCr)
Date: 02-2021
DOI: 10.1107/S2059798320016484
Abstract: Tannases are serine esterases that were first discovered in fungi more than one and half centuries ago. They catalyze the hydrolysis of the gallolyl ester bonds in gallotannins to release gallic acid, which is an important intermediate in the chemical and pharmaceutical industries. Since their discovery, fungal tannases have found wide industrial applications, although there is scarce knowledge about these enzymes at the molecular level, including their catalytic and substrate-binding sites. While this lack of knowledge hinders engineering efforts to modify the enzymes, many tannases have been isolated from various fungal strains in a search for the desired enzymatic properties. Here, the first crystal structure of a fungal tannase, that from Aspergillus niger , is reported. The enzyme possesses a typical α/β-hydrolase-fold domain with a large inserted cap domain, which together form a bowl-shaped hemispherical shape with a surface concavity surrounded by N-linked glycans. Gallic acid is bound at the junction of the two domains within the concavity by forming two hydrogen-bonding networks with neighbouring residues. One is formed around the carboxyl group of the gallic acid and involves residues from the hydrolase-fold domain, including those from the catalytic triad, which consists of Ser206, His485 and Asp439. The other is formed around the three hydroxyl groups of the compound, with the involvement of residues mainly from the cap domain, including Gln238, Gln239, His242 and Ser441. Gallic acid is bound in a sandwich-like mode by forming a hydrophobic contact with Ile442. All of these residues are found to be highly conserved among fungal and yeast tannases.
Publisher: Public Library of Science (PLoS)
Date: 06-03-2013
Publisher: International Union of Crystallography (IUCr)
Date: 29-05-2014
DOI: 10.1107/S1600576714009728
Abstract: Millions of crystallization trials are set up each year, with no clear metrics for determining if the experiments were correctly dispensed. This article reports the development of a software tool ( iQC – image Quality Control ) that recognizes factors associated with suboptimal experimental control during the setting up of protein crystallization trials. In its simplest form, iQC returns a report that gives an overall rating to the quality of an experimental setup. The iQC software is able to identify many common problems observed in setting up crystallization trials – droplets that have associated splatter droplets with air bubbles the positional accuracy of droplet placement elongated or otherwise `non-circular' drops – as well as detecting small and large droplets. An obvious use of this application is to track the status of the instrumentation used to set up crystallization trials in a multi-user laboratory.
Publisher: International Union of Crystallography (IUCr)
Date: 2018
DOI: 10.1107/S2053230X17017708
Abstract: Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus ( Ornithorhynchus anatinus ) MLP was determined at 1.82 Å resolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being observed ( P 1, C 2 and P 2 1 ). A 1.82 Å resolution native data set was collected from a crystal in space group P 1, with unit-cell parameters a = 51.2, b = 59.7, c = 63.1 Å, α = 80.15, β = 82.98, γ = 89.27°. The structure was solved by SAD phasing using a protein crystal derivatized with mercury in space group C 2, with unit-cell parameters a = 92.7, b = 73.2, c = 56.5 Å, β = 90.28°. MLP comprises a monomer of 12 helices and two short β-strands, with much of the N-terminus composed of loop regions. The crystal structure of MLP reveals no three-dimensional similarity to any known structures and reveals a heretofore unseen fold, supporting the idea that monotremes may be a rich source for the identification of novel proteins. It is hypothesized that MLP in monotreme milk has evolved to specifically support the unusual lactation strategy of this lineage and may have played a central role in the evolution of these mammals.
Publisher: Elsevier BV
Date: 05-2015
Publisher: International Union of Crystallography (IUCr)
Date: 24-03-2005
Publisher: American Chemical Society (ACS)
Date: 09-08-2010
DOI: 10.1021/JM100621S
Publisher: International Union of Crystallography (IUCr)
Date: 25-07-2014
DOI: 10.1107/S2053230X14016574
Abstract: Structural biology has contributed tremendous knowledge to the understanding of life on the molecular scale. The Protein Data Bank, a depository of this structural knowledge, currently contains over 100 000 protein structures, with the majority stemming from X-ray crystallography. As the name might suggest, crystallography requires crystals. As detectors become more sensitive and X-ray sources more intense, the notion of a crystal is gradually changing from one large enough to embellish expensive jewellery to objects that have external dimensions of the order of the wavelength of visible light. Identifying these crystals is a prerequisite to their study. This paper discusses developments in identifying these crystals during crystallization screening and distinguishing them from other potential outcomes. The practical aspects of ensuring that once a crystal is identified it can then be positioned in the X-ray beam for data collection are also addressed.
Publisher: International Union of Crystallography (IUCr)
Date: 29-10-2010
DOI: 10.1107/S0021889810040963
Abstract: The application of robotics to protein crystallization trials has resulted in the production of millions of images. Manual inspection of these images to find crystals and other interesting outcomes is a major rate-limiting step. As a result there has been intense activity in developing automated algorithms to analyse these images. The very first step for most systems that have been described in the literature is to delineate each droplet. Here, a novel approach that reaches over 97% success rate and subsecond processing times is presented. This will form the seed of a new high-throughput system to scrutinize massive crystallization c aigns automatically.
Publisher: Informa UK Limited
Date: 2011
Publisher: Public Library of Science (PLoS)
Date: 20-06-2018
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/CH14199
Abstract: This paper discusses the need for a systematic and standard naming nomenclature within the field of macromolecular crystallisation, and presents a set of rules and standard names which provides a start towards this end. The field of protein crystallisation is populated by biologists and chemists, and the dictionary in use needs to be unambiguous to both disciplines, yet must have useability as the most fundamental tenet if it is going to be widely adopted.
Publisher: Elsevier BV
Date: 11-2002
DOI: 10.1016/S0969-2126(02)00879-1
Abstract: Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site.
Publisher: American Chemical Society (ACS)
Date: 16-12-2014
DOI: 10.1021/CG4014569
Publisher: CSIRO Publishing
Date: 2013
DOI: 10.1071/CH13302
Abstract: The SAMPL (Statistical Assessment of the Modelling of Proteins and Ligands) challenge brought together experimentalists and modellers in an effort to improve our understanding of chemical and biochemical systems so better modelling tools can be developed. The most recent challenge, SAMPL3, held at Stanford University in August 2011, was an attempt to improve the methods used to predict how small fragment compounds bind to proteins, and the protein chosen for this test was bovine trypsin. Surface plasmon resonance was used to screen 500 compounds from a Maybridge fragment library and these compounds were subsequently used to soak crystals of trypsin and the best hits were also characterised by isothermal titration calorimetry. We present methods used for the surface plasmon resonance and the isothermal titration calorimetry experiments, as well as the results for these methods and those compounds that were found in the crystal structures.
Publisher: American Chemical Society (ACS)
Date: 10-05-2010
DOI: 10.1021/CG1004209
Publisher: American Society for Microbiology
Date: 07-2014
DOI: 10.1128/AEM.00916-14
Abstract: Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli . Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.
Publisher: Elsevier BV
Date: 02-2016
Publisher: International Union of Crystallography (IUCr)
Date: 27-06-2014
DOI: 10.1107/S2053230X1401262X
Abstract: While crystallization historically predates crystallography, it is a critical step for the crystallographic process. The rich history of crystallization and how that history influences current practices is described. The tremendous impact of crystallization screens on the field is discussed.
Publisher: International Union of Crystallography (IUCr)
Date: 11-2021
Publisher: Public Library of Science (PLoS)
Date: 24-03-2023
DOI: 10.1371/JOURNAL.PONE.0283124
Abstract: The use of imaging systems in protein crystallisation means that the experimental setups no longer require manual inspection to determine the outcome of the trials. However, it leads to the problem of how best to find images which contain useful information about the crystallisation experiments. The adoption of a deeplearning approach in 2018 enabled a four-class machine classification system of the images to exceed human accuracy for the first time. Underpinning this was the creation of a labelled training set which came from a consortium of several different laboratories. The MARCO classification model does not have the same accuracy on local data as it does on images from the original test set this can be somewhat mitigated by retraining the ML model and including local images. We have characterized the image data used in the original MARCO model, and performed extensive experiments to identify training settings most likely to enhance the local performance of a MARCO-dataset based ML classification model.
Publisher: MDPI AG
Date: 07-02-2020
Abstract: The process of macromolecular crystallisation almost always begins by setting up crystallisation trials using commercial or other premade screens, followed by cycles of optimisation where the crystallisation cocktails are focused towards a particular small region of chemical space. The screening process is relatively straightforward, but still requires an understanding of the plethora of commercially available screens. Optimisation is complicated by requiring both the design and preparation of the appropriate secondary screens. Software has been developed in the C3 lab to aid the process of choosing initial screens, to analyse the results of the initial trials, and to design and describe how to prepare optimisation screens.
Publisher: International Union of Crystallography (IUCr)
Date: 25-11-2020
DOI: 10.1107/S2053230X20015216
Abstract: Ssr4 is a yeast protein from Schizosaccharomyces pombe and is an essential part of the chromatin-remodelling [SWI/SNF and RSC (remodelling the structure of chromatin)] complexes found in S. pombe . These complexes (or their homologues) regulate gene expression in eukaryotic organisms, affecting a large number of genes both positively and negatively. The downstream effects are seen in development, and in humans have implications for disease such as cancer. The chromatin structure is altered by modifying the DNA–histone contacts, thus opening up or closing down sections of DNA to specific transcription factors that regulate the transcription of genes. The Ssr4 sequence has little homology to other sequences in the Protein Data Bank, so the structure was solved using an iodine derivative with SAD phasing. The structure of the N-terminal domain is an antiparallel β-sheet of seven strands with α-helices on one side and random coil on the other. The structure is significantly different to deposited structures and was used as a target in the most recent Critical Assessment of Techniques for Protein Structure Prediction (CASP predictioncenter.org/) competition.
Publisher: International Union of Crystallography (IUCr)
Date: 12-2021
Publisher: International Union of Crystallography (IUCr)
Date: 21-02-2019
DOI: 10.1107/S2053230X19000141
Abstract: Crystallization is in many cases a critical step for solving the three-dimensional structure of a protein molecule. Determining which set of chemicals to use in the initial screen is typically agnostic of the protein under investigation however, crystallization efficiency could potentially be improved if this were not the case. Previous work has assumed that sequence similarity may provide useful information about appropriate crystallization cocktails however, the authors are not aware of any quantitative verification of this assumption. This research investigates whether, given current information, one can detect any correlation between sequence similarity and crystallization cocktails. BLAST was used to quantitate the similarity between protein sequences in the Protein Data Bank, and this was compared with three estimations of the chemical similarities of the respective crystallization cocktails. No correlation was detected between proteins of similar (but not identical) sequence and their crystallization cocktails, suggesting that methods of determining screens based on this assumption are unlikely to result in screens that are better than those currently in use.
Publisher: Elsevier BV
Date: 05-2018
Publisher: American Society for Microbiology
Date: 05-2017
DOI: 10.1128/AEM.03365-16
Abstract: The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na + or Mg 2+ , analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity. IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.
Publisher: American Chemical Society (ACS)
Date: 23-03-2012
DOI: 10.1021/CO2001718
Abstract: A protocol is presented for the high-throughput (HT) production of lyotropic liquid crystalline phases from libraries of lipids and lipid mixtures using standard liquid dispensing robotics, implementing methods that circumvent the problems traditionally associated with handling the highly viscous cubic phase. In addition, the ability to structurally characterize lipidic phases and assess functionality for membrane proteins contained within cubic phases, in a HT manner, is demonstrated. The techniques are combined and exemplified using the application of membrane protein crystallization within lipidic cubic phases.
Publisher: Wiley
Date: 16-11-2015
DOI: 10.1002/PROT.24942
Publisher: Wiley
Date: 17-08-2011
Abstract: An optimised method of solution cyclisation gave us access to a series of peptides including SLKIDNLD (2). We investigated the crystallographic complexes of the HIV integrase (HIV-IN) catalytic core domain with 13 of the peptides and identified multiple interactions at the binding site, including hydrogen bonds with residues Thr125 and Gln95, that have not previously been described as being accessible within the binding site. We show that the peptides inhibit the interaction of lens epithelium-derived growth factor (LEDGF) with HIV-IN in a proximity AlphaScreen assay and in an assay for the LEDGF enhancement of HIV-IN strand transfer. The interactions identified represent a potential framework for the development of new HIV-IN inhibitors.
Publisher: International Union of Crystallography (IUCr)
Date: 12-2021
Publisher: International Union of Crystallography (IUCr)
Date: 31-01-2014
DOI: 10.1107/S1399004713031052
Abstract: The X-ray crystal structure of the complex of protein tyrosine phosphatase 1B with nitrate anion has been determined and modelled quantum-mechanically. Two protomers were present in the structure, one with the mechanistically important WPD loop closed and the other with this loop open. Nitrate was observed bound to each protomer, making close contacts with the S atom of the catalytic cysteine and a tyrosine residue from a crystallographically related protomer.
Publisher: International Union of Crystallography (IUCr)
Date: 16-12-2021
Publisher: Springer Science and Business Media LLC
Date: 13-10-2016
DOI: 10.1038/SREP35198
Abstract: Chemoreceptors enable bacteria to detect chemical signals in the environment and navigate towards niches that are favourable for survival. The sensor domains of chemoreceptors function as the input modules for chemotaxis systems, and provide sensory specificity by binding specific ligands. Cache-like domains are the most common extracellular sensor module in prokaryotes, however only a handful have been functionally or structurally characterised. Here, we have characterised a chemoreceptor Cache-like sensor domain (PscD-SD) from the plant pathogen Pseudomonas syringae pv. actinidiae ( Psa ). High-throughput fluorescence thermal shift assays, combined with isothermal thermal titration calorimetry, revealed that PscD-SD binds specifically to C 2 (glycolate and acetate) and C 3 (propionate and pyruvate) carboxylates. We solved the structure of PscD-SD in complex with propionate using X-ray crystallography. The structure reveals the key residues that comprise the ligand binding pocket and dictate the specificity of this sensor domain for C 2 and C 3 carboxylates. We also demonstrate that all four carboxylate ligands are chemoattractants for Psa , but only two of these (acetate and pyruvate) are utilisable carbon sources. This result suggests that in addition to guiding the bacteria towards nutrients, another possible role for carboxylate sensing is in locating potential sites of entry into the host plant.
Publisher: Public Library of Science (PLoS)
Date: 24-07-2013
DOI: 10.1371/ANNOTATION/EB238F0F-7582-4318-AF95-64AC98D3B0BE
Publisher: International Union of Crystallography (IUCr)
Date: 21-10-2002
DOI: 10.1107/S0907444902016852
Abstract: In contrast to academic pursuits of structural genomics, Structural GenomiX (SGX) solves protein structures at high throughput for the main purpose of enhancing drug-discovery projects, either internally or in partnership with pharmaceutical/biotechnology companies. This involves a radical redesign of the pipeline of methods that turn a gene sequence into a three-dimensional protein structure. The various processes all report electronically to a Laboratory Information Management System (LIMS) to make sure all the parameters of the experiment are recorded in an accessible and `mineable' form, helping guarantee reproducibility of results. Quality control at several key points keeps the process from branching out on a wrong hypothesis. Protein annotation, in a broad sense, takes care of the interpretation of a protein crystal structure or the crystal structure of one or several protein–ligand complexes. This interpretation both gathers all necessary biological information (protein function, mechanism, specific features within a protein family etc .) and hands over this information in a form accessible to medicinal chemistry teams designing specific small-molecule agonists or antagonists.
Publisher: International Union of Crystallography (IUCr)
Date: 30-04-2019
Publisher: AIP
Date: 2013
DOI: 10.1063/1.4825019
Publisher: International Union of Crystallography (IUCr)
Date: 14-02-2009
Publisher: Cold Spring Harbor Laboratory
Date: 28-09-2022
DOI: 10.1101/2022.09.28.509867
Abstract: The use of imaging systems in protein crystallisation means that the experimental setups no longer require manual inspection to determine the outcome of the trials. However, it leads to the problem of how best to find images which contain useful information about the crystallisation experiments. The adoption of a deeplearning approach in 2018 enabled a four-class machine classification system of the images to exceed human accuracy for the first time. Underpinning this was the creation of a labelled training set which came from a consortium of several different laboratories. The MARCO classification model does not have the same accuracy on local data as it does on images from the original test set this can be somewhat mitigated by retraining the ML model and including local images. We have characterized the image data used in the original MARCO model, and performed extensive experiments to identify training settings most likely to enhance the local performance of a MARCO-dataset based ML classification model.
Publisher: International Union of Crystallography (IUCr)
Date: 23-09-2015
DOI: 10.1107/S2053230X15012662
Abstract: There is strong evidence to suggest that a protein s le needs to be well folded and uniform in order to form protein crystals, and it is accepted knowledge that the formulation can have profound effects on the behaviour of the protein s le. The technique of differential scanning fluorimetry (DSF) is a very accessible method to determine protein stability as a function of the formulation chemistry and the temperature. A erse set of 252 soluble protein s les was subjected to a standard formulation-screening protocol using DSF. Automated analysis of the DSF results suggest that in over 35% of cases buffer screening significantly increases the stability of the protein s le. Of the 28 standard formulations tested, three stood out as being statistically better than the others: these included a formulation containing the buffer citrate, long known to be `protein friendly' bis-tris and ADA were also identified as being very useful buffers in protein formulations.
Publisher: International Union of Crystallography (IUCr)
Date: 25-02-2004
Publisher: International Union of Crystallography (IUCr)
Date: 19-02-2014
Publisher: International Union of Crystallography (IUCr)
Date: 26-06-2018
DOI: 10.1107/S2053230X18008038
Abstract: The process of producing suitable crystals for X-ray diffraction analysis most often involves the setting up of hundreds (or thousands) of in idual crystallization trials, each of which must be repeatedly examined for crystals or hints of crystallinity. Currently, the only real way to address this bottleneck is to use an automated imager to capture images of the trials. However, the images still need to be assessed for crystals or other outcomes. Ideally, there would exist some rapid and reliable machine-analysis tool to translate the images into a quantitative result. However, as yet no such tool exists in wide usage, despite this being a well recognized problem. One of the issues in creating robust automatic image-analysis software is the lack of reliable data for training machine-learning algorithms. Here, a mobile application, Cinder , has been developed which allows crystallization images to be scored quickly on a smartphone or tablet. The Cinder scores are inserted into the appropriate table in a crystallization database and are immediately available to the user through a more sophisticated web interface, allowing more detailed analyses. A sharp increase in the number of scored images was observed after Cinder was released, which in turn provides more data for training machine-learning tools.
Publisher: International Union of Crystallography (IUCr)
Date: 02-2018
DOI: 10.1107/S1600576717016727
Abstract: Managing chemical stocks and s les in any laboratory is an arduous task in a crystallization laboratory this is particularly burdensome, given the need for many stocks to facilitate optimization of crystal hits obtained from screening experiments. Although inventory management is widespread in retail and other arenas, most small academic laboratories do not adopt formal stock management systems. Without an overarching system for handling stocks and s les, problems such as stock duplication, inappropriate stock storage and insufficient labelling are rife. Two applications have been developed in the Collaborative Crystallization Centre, the first of which manages the hundreds of stocks used for crystallization, and a second which manages protein (and other) s les stored in the 193 K freezer. Both applications are built around a simple database, with a Python front end that allows s les or stocks to be scanned in or out. Information from a decade of crystallization stock usage allows a good estimation of what chemicals are used (and in what quantities) in a crystallization laboratory.
Publisher: International Union of Crystallography (IUCr)
Date: 17-08-2020
DOI: 10.1107/S2059798320010505
Abstract: Cancer is one of the leading causes of mortality in humans, and recent work has focused on the area of immuno-oncology, in which the immune system is used to specifically target cancerous cells. Ectonucleotide pyrophosphatase hosphodiesterase 1 (ENPP1) is an emerging therapeutic target in human cancers owing to its role in degrading cyclic GMP-AMP (cGAMP), an agonist of the stimulator of interferon genes (STING). The available structures of ENPP1 are of the mouse enzyme, and no structures are available with anything other than native nucleotides. Here, the first X-ray crystal structures of the human ENPP1 enzyme in an apo form, with bound nucleotides and with two known inhibitors are presented. The availability of these structures and a robust crystallization system will allow the development of structure-based drug-design c aigns against this attractive cancer therapeutic target.
Publisher: EMBO
Date: 26-08-2020
Publisher: Public Library of Science (PLoS)
Date: 06-11-2018
Publisher: Springer International Publishing
Date: 2019
Publisher: International Union of Crystallography (IUCr)
Date: 29-04-2019
Publisher: AIP Publishing
Date: 11-2019
DOI: 10.1063/1.5122849
Abstract: The WD40-repeat protein WDR5 scaffolds various epigenetic writers and is a critical component of the mammalian SET/MLL histone methyltransferase complex. Dysregulation of the MLL1 catalytic function is associated with mixed-lineage leukemia, and antagonism of the WDR5-MLL1 interaction by small molecules has been proposed as a therapeutic strategy for MLL-rearranged cancers. Small molecule binders of the “WIN” site of WDR5 that cause displacement from chromatin have been additionally implicated to be of broader use in cancer treatment. In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-containing compound that is bound in the WIN site. The subsequent medicinal chemistry c aign—guided by a suite of high-resolution cocrystal structures with WDR5—progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine a tripeptide containing one such substitution was resolved in a high resolution structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (kd = ∼0.06 s−1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics.
Publisher: Public Library of Science (PLoS)
Date: 21-09-2015
Publisher: International Union of Crystallography (IUCr)
Date: 29-02-2008
Publisher: Public Library of Science (PLoS)
Date: 09-02-2018
Publisher: International Union of Crystallography (IUCr)
Date: 23-12-2011
Publisher: American Chemical Society (ACS)
Date: 27-10-2000
DOI: 10.1021/BI001539C
Abstract: The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.
Publisher: International Union of Crystallography (IUCr)
Date: 30-09-2014
DOI: 10.1107/S2053230X1401841X
Abstract: The REMARK280 field of the Protein Data Bank is the richest open source of successful crystallization information. The REMARK280 field is optional and currently uncurated, so significant effort needs to be applied to extract reliable data. There are well over 15 000 crystallization conditions available commercially from 12 different vendors. After putting the PDB crystallization information and the commercial cocktail data into a consistent format, these data are used to extract information about the overlap between the two sets of crystallization conditions. An estimation is made as to which commercially available conditions are most appropriate for producing well diffracting crystals by looking at which commercial conditions are found unchanged (or almost unchanged) in the PDB. Further analyses include which commercial kits are the most appropriate for shotgun or more traditional approaches to crystallization screening. This analysis suggests that almost 40% of the crystallization conditions found currently in the PDB are identical or very similar to a commercial condition.
Publisher: International Union of Crystallography (IUCr)
Date: 2017
DOI: 10.1107/S2053230X16020008
Abstract: The NAD-dependent malonate-semialdehyde dehydrogenase KES23460 from Pseudomonas sp. strain AAC makes up half of a bicistronic operon responsible for β-alanine catabolism to produce acetyl-CoA. The KES23460 protein has been heterologously expressed, purified and used to generate crystals suitable for X-ray diffraction studies. The crystals belonged to space group P 2 1 2 1 2 1 and diffracted X-rays to beyond 3 Å resolution using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4zz7 as the search model.
Publisher: International Union of Crystallography (IUCr)
Date: 2017
DOI: 10.1107/S2053230X16019658
Abstract: The putrescine aminotransferase KES24511 from Pseudomonas sp. strain AAC was previously identified as an industrially relevant enzyme based on the discovery that it is able to promiscuously catalyse the transamination of 12-aminododecanoic acid. Here, the cloning, heterologous expression, purification and successful crystallization of the KES24511 protein are reported, which ultimately generated crystals adopting space group I 2. The crystals diffracted X-rays to 2.07 Å resolution and data were collected using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4a6t as the search model.
Publisher: International Union of Crystallography (IUCr)
Date: 28-11-2014
DOI: 10.1107/S1399004714022767
Abstract: Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B 5 ) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein–protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface.
Publisher: International Union of Crystallography (IUCr)
Date: 26-08-2009
Publisher: Cold Spring Harbor Laboratory
Date: 19-06-2020
DOI: 10.1101/2020.06.18.160614
Abstract: Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications 1,2 . Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses 3 . We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.
Publisher: International Union of Crystallography (IUCr)
Date: 26-02-2015
DOI: 10.1107/S1399004715000619
Abstract: Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organism Pseudomonas sp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role ( i.e. atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit.
Publisher: Cold Spring Harbor Laboratory
Date: 12-08-2021
DOI: 10.1101/2021.08.11.456002
Abstract: Protein crystallisation has for decades been a critical and restrictive step in macro-molecular structure determination via X-ray diffraction. Crystallisation typically involves a multi-stage exploration of the available chemical space, beginning with an initial s ling (screening) followed by iterative refinement (optimisation). Effective screening is important for reducing the number of optimisation rounds required, reducing the cost and time required to determine a structure. Here, we propose an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) and compare it with the previously derived (2014) Shotgun I screen. In an update to that analysis, we clarify that the Shotgun approach entails finding the crystallisation conditions which cover the most erse space of proteins by sequence found in the PDB - which can be mapped to the well known Maximum Coverage problem in computer science. With this realisation we are able to apply a more effective algorithm for selecting conditions, such that the Shotgun II screen outperforms the Shotgun I screen both in protein coverage and quantity of data input. Our data demonstrates that the Shotgun I screen, compared with alternatives, has been remarkably successful over the seven years it has been in use, indicating that Shotgun II is likely to be a highly effective screen.
Publisher: International Union of Crystallography (IUCr)
Date: 26-06-2019
DOI: 10.1107/S2059798319008131
Abstract: The structure of BgaR, a transcriptional regulator of the lactose operon in Clostridium perfringens , has been solved by SAD phasing using a mercury derivative. BgaR is an exquisite sensor of lactose, with a binding affinity in the low-micromolar range. This sensor and regulator has been captured bound to lactose and to lactulose as well as in a nominal apo form, and was compared with AraC, another saccharide-binding transcriptional regulator. It is shown that the saccharides bind in the N-terminal region of a jelly-roll fold, but that part of the saccharide is exposed to bulk solvent. This differs from the classical AraC saccharide-binding site, which is mostly sequestered from the bulk solvent. The structures of BgaR bound to lactose and to lactulose highlight how specific and nonspecific interactions lead to a higher binding affinity of BgaR for lactose compared with lactulose. Moreover, solving multiple structures of BgaR in different space groups, both bound to saccharides and unbound, verified that the dimer interface along a C-terminal helix is similar to the dimer interface observed in AraC.
Publisher: Elsevier BV
Date: 12-2009
Abstract: To provide an experimental basis for a comprehensive molecular modeling evaluation study, 500 fragments from the Maybridge fragment library were soaked into crystals of bovine pancreatic trypsin and the structures determined by X-ray crystallography. The soaking experiments were performed in both single and pooled aliquots to determine if combination of fragments is an appropriate strategy. A further set of data was obtained from co-crystallizing the pooled fragments with the protein. X-ray diffraction data were collected on approximately 1000 crystals at the Australian Synchrotron, and these data were subsequently processed, and the preliminary analysis was performed with a custom software application (Jigsaw), which combines available software packages for structure solution and analysis.
Publisher: Wiley
Date: 13-01-2010
DOI: 10.1002/PRO.312
Publisher: Oxford University Press (OUP)
Date: 11-2012
Publisher: American Chemical Society (ACS)
Date: 12-11-1999
DOI: 10.1021/BI9913855
Abstract: The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7CC00846E
Abstract: Novel X-ray crystal structures of cyclic d / l peptide nanotubes in antiparallel and parallel configurations.
Publisher: Wiley
Date: 20-05-2013
DOI: 10.1111/MMI.12249
Publisher: Springer Science and Business Media LLC
Date: 08-12-2021
DOI: 10.1038/S41467-021-27184-W
Abstract: Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.
Publisher: IEEE
Date: 12-2011
Publisher: International Union of Crystallography (IUCr)
Date: 09-2020
Publisher: International Union of Crystallography (IUCr)
Date: 11-2019
Publisher: Public Library of Science (PLoS)
Date: 10-07-2012
Publisher: Public Library of Science (PLoS)
Date: 31-10-2007
Publisher: Springer Science and Business Media LLC
Date: 16-02-2014
Publisher: International Union of Crystallography (IUCr)
Date: 14-12-2005
Publisher: International Society for Horticultural Science (ISHS)
Date: 04-2017
Publisher: Elsevier BV
Date: 10-1998
Abstract: Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression to facilitate protein folding by including zinc and reducing the induction temperature. Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies.
Publisher: International Union of Crystallography (IUCr)
Date: 27-10-2021
DOI: 10.1107/S2059798321009724
Abstract: Protein crystallization has for decades been a critical and restrictive step in macromolecular structure determination via X-ray diffraction. Crystallization typically involves a multi-stage exploration of the available chemical space, beginning with an initial s ling (screening) followed by iterative refinement (optimization). Effective screening is important for reducing the number of optimization rounds required, reducing the cost and time required to determine a structure. Here, an initial screen (Shotgun II) derived from analysis of the up-to-date Protein Data Bank (PDB) is proposed and compared with the previously derived (2014) Shotgun I screen. In an update to that analysis, it is clarified that the Shotgun approach entails finding the crystallization conditions that cover the most erse space of proteins by sequence found in the PDB, which can be mapped to the well known maximum coverage problem in computer science. With this realization, it was possible to apply a more effective algorithm for selecting conditions. In-house data demonstrate that compared with alternatives, the Shotgun I screen has been remarkably successful over the seven years that it has been in use, indicating that Shotgun II is also likely to be a highly effective screen.
Publisher: International Union of Crystallography (IUCr)
Date: 31-10-2008
Publisher: SAGE Publications
Date: 12-2002
Publisher: International Union of Crystallography (IUCr)
Date: 08-2019
Publisher: Springer Science and Business Media LLC
Date: 21-12-2015
DOI: 10.1038/NCOMMS10278
Abstract: Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na + hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated.
Publisher: International Union of Crystallography (IUCr)
Date: 30-04-2019
Publisher: International Union of Crystallography (IUCr)
Date: 25-11-2011
DOI: 10.1107/S1744309111038395
Abstract: Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS from the grapevine Vitis vinifera (Vv-DHDPS). Following in-drop cleavage of the hexahistidine tag, cocrystals of Vv-DHDPS with the substrate pyruvate were grown in 0.1 M Bis-Tris propane pH 8.2, 0.2 M sodium bromide, 20%( w / v ) PEG 3350. X-ray diffraction data in space group P 1 at a resolution of 2.2 Å are presented. Preliminary diffraction data analysis indicated the presence of eight molecules per asymmetric unit ( V M = 2.55 Å 3 Da −1 , 52% solvent content). The pending crystal structure of Vv-DHDPS will provide insight into the molecular evolution in quaternary structure of DHDPS enzymes.
Location: Australia
No related grants have been discovered for Janet Newman.