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Instruments / Technical Area	Publication Details	Published
	Helical ensembles outperform ideal helices in molecular replacement Filomeno Sanchez Rodriguez , Adam J. Simpkin , Owen R. Davies , Ronan M. Keegan , Daniel J. Rigden Acta Crystallographica Section D Structural Biology 76 DOI: 10.1107/S205979832001133X Open Access Show Abstract ▼ 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.	Oct 2020
I04-Macromolecular Crystallography	SIMBAD : a sequence-independent molecular-replacement pipeline 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 Acta Crystallographica Section D Structural Biology 74 DOI: 10.1107/S2059798318005752 Diamond Proposal Number(s): [15945] Open Access Show Abstract ▼ 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.	Jul 2018
I03-Macromolecular Crystallography	Structure and function of the type III pullulan hydrolase from Thermococcus kodakarensis Jingxu Guo , Alun Coker , Steve P. Wood , Jonathan Cooper , Ronan M. Keegan , Nasir Ahmad , Majida Atta Muhammad , Naeem Rashid , Muhummad Akhtar Acta Crystallographica Section D Structural Biology 74, 305 - 314 DOI: 10.1107/S2059798318001754 Diamond Proposal Number(s): [12342] Show Abstract ▼ 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.	Apr 2018
	CCP 4 i 2: the new graphical user interface to the CCP 4 program suite 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 Acta Crystallographica Section D Structural Biology 74, 68 - 84 DOI: 10.1107/S2059798317016035 Open Access Show Abstract ▼ 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.	Feb 2018
I03-Macromolecular Crystallography	Structure of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP Ronan Keegan , Andrey Lebedev , Peter Erskine , Jingxu Guo , S. P. Wood , D. J. Hopper , S. E. J. Rigby , J. B. Cooper Acta Crystallographica Section D Biological Crystallography 70, 2444 - 2454 DOI: 10.1107/S1399004714015053 PMID: 25195757 Diamond Proposal Number(s): [8922, 1425, 7131] Open Access Show Abstract ▼ 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.	Sep 2014
NONE-No attached Diamond beamline	CCP4 web services Ville Uski , Charles Ballard , Ronan Keegan , Eugene Krissinel , Andrey Lebedev , David Waterman , Marcin Wojdyr Show Abstract ▼ 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.	Aug 2014
I02-Macromolecular Crystallography	Crystallization and preliminary X-ray characterization of the 2,4′-dihydroxyacetophenone dioxygenase from 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 Acta Crystallographica Section F Structural Biology Communications 70, 823 - 826 DOI: 10.1107/S2053230X14009649 PMID: 24915102 Diamond Proposal Number(s): [1425, 7131] Show Abstract ▼ 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.	Jun 2014
	Evaluating the solution from MrBUMP and BALBES Ronan Keegan , Fei Long , Vincent J. Fazio , Martyn Winn , Garib N. Murshudov , Alexei A. Vagin Acta Crystallographica Section D Biological Crystallography 67 (4), 313 - 323 DOI: 10.1107/S0907444911007530 Open Access Show Abstract ▼ Abstract: Molecular replacement is one of the key methods used to solve the problem of determining the phases of structure factors in protein structure solution from X-ray image diffraction data. Its success rate has been steadily improving with the development of improved software methods and the increasing number of structures available in the PDB for use as search models. Despite this, in cases where there is low sequence identity between the target-structure sequence and that of its set of possible homologues it can be a difficult and time-consuming chore to isolate and prepare the best search model for molecular replacement. MrBUMP and BALBES are two recent developments from CCP4 that have been designed to automate and speed up the process of determining and preparing the best search models and putting them through molecular replacement. Their intention is to provide the user with a broad set of results using many search models and to highlight the best of these for further processing. An overview of both programs is presented along with a description of how best to use them, citing case studies and the results of large-scale testing of the software.	Apr 2011

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