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Open Access
Abstract: Determination of protein structures typically entails building a model that satisfies the collected experimental observations and its deposition in the Protein Data Bank. Experimental limitations can lead to unavoidable uncertainties during the process of model building, which result in the introduction of errors into the deposited model. Many metrics are available for model validation, but most are limited to consideration of the physico-chemical aspects of the model or its match to the experimental data. The latest advances in the field of deep learning have enabled the increasingly accurate prediction of inter-residue distances, an advance which has played a pivotal role in the recent improvements observed in the field of protein ab initio modelling. Here, new validation methods are presented based on the use of these precise inter-residue distance predictions, which are compared with the distances observed in the protein model. Sequence-register errors are particularly clearly detected and the register shifts required for their correction can be reliably determined. The method is available in the ConKit package (https://www.conkit.org).
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Dec 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12342]
Abstract: Insect juvenile hormones (JHs) are a family of sesquiterpenoid molecules that are secreted into the haemolymph. JHs have multiple roles in insect development, metamorphosis and sexual maturation. A number of pesticides work by chemically mimicking JHs, thus preventing insects from developing and reproducing normally. The haemolymph levels of JH are governed by the rates of its biosynthesis and degradation. One enzyme involved in JH catabolism is JH diol kinase (JHDK), which uses ATP (or GTP) to phosphorylate JH diol to JH diol phosphate, which can be excreted. The X-ray structure of JHDK from the silkworm Bombyx mori has been determined at a resolution of 2.0 Å with an R factor of 19.0% and an Rfree of 24.8%. The structure possesses three EF-hand motifs which are occupied by calcium ions. This is in contrast to the recently reported structure of the JHDK-like-2 protein from B. mori (PDB entry 6kth), which possessed only one calcium ion. Since JHDK is known to be inhibited by calcium ions, it is likely that our structure represents the calcium-inhibited form of the enzyme. The electrostatic surface of the protein suggests a binding site for the triphosphate of ATP close to the N-terminal end of the molecule in a cavity between the N- and C-terminal domains. Superposition with a number of calcium-activated photoproteins suggests that there may be parallels between the binding of JH diol to JHDK and the binding of luciferin to aequorin.
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Dec 2021
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Open Access
Abstract: Covariance-based predictions of residue contacts and inter-residue distances are an increasingly popular data type in protein bioinformatics. Here we present ConPlot, a web-based application for convenient display and analysis of contact maps and distograms. Integration of predicted contact data with other predictions is often required to facilitate inference of structural features. ConPlot can therefore use the empty space near the contact map diagonal to display multiple coloured tracks representing other sequence-based predictions. Popular file formats are natively read and bespoke data can also be flexibly displayed. This novel visualisation will enable easier interpretation of predicted contact maps.
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Jan 2021
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Open Access
Abstract: The conventional approach in molecular replacement is the use of a related structure as a search model. However, this is not always possible as the availability of such structures can be scarce for poorly characterized families of proteins. In these cases, alternative approaches can be explored, such as the use of small ideal fragments that share high, albeit local, structural similarity with the unknown protein. Earlier versions of AMPLE enabled the trialling of a library of ideal helices, which worked well for largely helical proteins at suitable resolutions. Here, the performance of libraries of helical ensembles created by clustering helical segments is explored. The impacts of different B-factor treatments and different degrees of structural heterogeneity are explored. A 30% increase in the number of solutions obtained by AMPLE was observed when using this new set of ensembles compared with the performance with ideal helices. The boost in performance was notable across three different fold classes: transmembrane, globular and coiled-coil structures. Furthermore, the increased effectiveness of these ensembles was coupled to a reduction in the time required by AMPLE to reach a solution. AMPLE users can now take full advantage of this new library of search models by activating the `helical ensembles' mode.
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Oct 2020
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I04-Macromolecular Crystallography
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Adam J.
Simpkin
,
Felix
Simkovic
,
Jens M. H.
Thomas
,
Martin
Savko
,
Andrey
Lebedev
,
Ville
Uski
,
Charles
Ballard
,
Marcin
Wojdyr
,
Rui
Wu
,
Ruslan
Sanishvili
,
Yibin
Xu
,
María-Natalia
Lisa
,
Alejandro
Buschiazzo
,
William
Shepard
,
Daniel J.
Rigden
,
Ronan M.
Keegan
Diamond Proposal Number(s):
[15945]
Open Access
Abstract: The conventional approach to finding structurally similar search models for use in molecular replacement (MR) is to use the sequence of the target to search against those of a set of known structures. Sequence similarity often correlates with structure similarity. Given sufficient similarity, a known structure correctly positioned in the target cell by the MR process can provide an approximation to the unknown phases of the target. An alternative approach to identifying homologous structures suitable for MR is to exploit the measured data directly, comparing the lattice parameters or the experimentally derived structure-factor amplitudes with those of known structures. Here, SIMBAD, a new sequence-independent MR pipeline which implements these approaches, is presented. SIMBAD can identify cases of contaminant crystallization and other mishaps such as mistaken identity (swapped crystallization trays), as well as solving unsequenced targets and providing a brute-force approach where sequence-dependent search-model identification may be nontrivial, for example because of conformational diversity among identifiable homologues. The program implements a three-step pipeline to efficiently identify a suitable search model in a database of known structures. The first step performs a lattice-parameter search against the entire Protein Data Bank (PDB), rapidly determining whether or not a homologue exists in the same crystal form. The second step is designed to screen the target data for the presence of a crystallized contaminant, a not uncommon occurrence in macromolecular crystallography. Solving structures with MR in such cases can remain problematic for many years, since the search models, which are assumed to be similar to the structure of interest, are not necessarily related to the structures that have actually crystallized. To cater for this eventuality, SIMBAD rapidly screens the data against a database of known contaminant structures. Where the first two steps fail to yield a solution, a final step in SIMBAD can be invoked to perform a brute-force search of a nonredundant PDB database provided by the MoRDa MR software. Through early-access usage of SIMBAD, this approach has solved novel cases that have otherwise proved difficult to solve.
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Jul 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12342]
Abstract: Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase from Thermococcus kodakarensis (TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described. TK-PUL shows highest activity in the temperature range 95–100°C and has a pH optimum in the range 3.5–4.2. Its unique ability to hydrolyse maltotriose into maltose and glucose has not been reported for other homologous enzymes. The crystal structure of TK-PUL has been determined at a resolution of 2.8 Å and represents the first analysis of a type III pullulan hydrolyse. The structure reveals that the last part of the N-terminal domain and the C-terminal domain are significantly different from homologous structures. In addition, the loop regions at the active-site end of the central catalytic domain are quite different. The enzyme has a well defined calcium-binding site and possesses a rare vicinal disulfide bridge. The thermostability of TK-PUL and its homologues may be attributable to several factors, including the increased content of salt bridges, helical segments, Pro, Arg and Tyr residues and the decreased content of serine.
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Apr 2018
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Liz
Potterton
,
Jon
Agirre
,
Charles
Ballard
,
Kevin
Cowtan
,
Eleanor
Dodson
,
Phil R.
Evans
,
Huw T.
Jenkins
,
Ronan
Keegan
,
Eugene
Krissinel
,
Kyle
Stevenson
,
Andrey
Lebedev
,
Stuart J.
Mcnicholas
,
Robert A.
Nicholls
,
Martin
Noble
,
Navraj S.
Pannu
,
Christian
Roth
,
George
Sheldrick
,
Pavol
Skubak
,
Johan
Turkenburg
,
Ville
Uski
,
Frank
Von Delft
,
David
Waterman
,
Keith
Wilson
,
Martyn
Winn
,
Marcin
Wojdyr
Open Access
Abstract: The CCP4 (Collaborative Computational Project, Number 4) software suite for macromolecular structure determination by X-ray crystallography groups brings together many programs and libraries that, by means of well established conventions, interoperate effectively without adhering to strict design guidelines. Because of this inherent flexibility, users are often presented with diverse, even divergent, choices for solving every type of problem. Recently, CCP4 introduced CCP4i2, a modern graphical interface designed to help structural biologists to navigate the process of structure determination, with an emphasis on pipelining and the streamlined presentation of results. In addition, CCP4i2 provides a framework for writing structure-solution scripts that can be built up incrementally to create increasingly automatic procedures.
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Feb 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8922, 1425, 7131]
Open Access
Abstract: The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the -hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis.
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Sep 2014
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NONE-No attached Diamond beamline
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Abstract: The CCP4 software suite [1] provides a comprehensive set of tools for use in the macromolecule structure solution process by X-ray
crystallography. Traditionally, these tools have been run through the graphical interface or the command line on each user's personal workstation. Recently, some of the tools, including the molecular replacement pipelines Balbes [2] and MrBUMP [3] have been provided as web services in the Research Complex at Harwell. These pipelines can be especially useful in cases where there is low
sequence identity between the target-structure sequence and that of its set of possible homologues. These services can be accessed
through a web client, allowing one to submit molecular replacement jobs to our Linux cluster and view the results from these jobs. The molecular replacement pipelines are ideal candidates for web services, as they require installation and maintenance of large databases and benefit from parallel computing resources, provided by the cluster. Further plans for web services will be discussed. With ever-increasing mobility of scientific setups and the ubiquity of ultra-portable devices, there is a demand for a consistent
framework of remote crystallographic computations and data maintenance. This framework is planned to include an interface for synchronising data with the facilities of Diamond Light Source, as well as with local CCP4 GUI-2 setups.
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Aug 2014
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I02-Macromolecular Crystallography
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G.
Beaven
,
A.
Bowyer
,
P.
Erskine
,
S. P.
Wood
,
A.
Mccoy
,
L.
Coates
,
R.
Keegan
,
A.
Lebedev
,
D. J.
Hopper
,
M. A.
Kaderbhai
,
J. B.
Cooper
Diamond Proposal Number(s):
[1425, 7131]
Abstract: The enzyme 2,4'-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits each containing nonhaem iron and its sequence suggests that it belongs to the cupin family of dioxygenases. By the use of limited chymotrypsinolysis, the DAD enzyme from Alcaligenes sp. 4HAP has been crystallized in a form that diffracts synchrotron radiation to a resolution of 2.2 Å.
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Jun 2014
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