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Instruments / Technical Area	Publication Details	Published
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila Jason S. Wilson , Alicia M. Churchill-angus , Simon P. Davies , Svetlana E. Sedelnikova , Svetomir B. Tzokov , John B. Rafferty , Per A. Bullough , Claudine Bisson , Patrick J. Baker Nature Communications 10, 2900 DOI: 10.1038/s41467-019-10777-x Diamond Proposal Number(s): [8987, 12788, 17773] Open Access Show Abstract ▼ Abstract: The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.	Jul 2019
I02-Macromolecular Crystallography	A new mechanism for high-affinity uptake of C4-dicarboxylates in bacteria revealed by the structure of rhodopseudomonas palustris matc (RPA3494), a periplasmic binding-protein of the tripartite tricarboxylate transporter (TTT) family Leonardo T. Rosa , Samuel R. Dix , John B. Rafferty , David J. Kelly Journal Of Molecular Biology DOI: 10.1016/j.jmb.2018.11.016 Diamond Proposal Number(s): [12788] Open Access Show Abstract ▼ Abstract: C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric-acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the Tripartite Tricarboxylate Transport (TTT) family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to L- and D-malate with Kd values of 27 and 21 nM respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd 110 nM) and fumarate (Kd 400 nM). The 2.1 Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data, and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized TTT family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.	Nov 2018
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for high-affinity adipate binding to AdpC (RPA4515), an orphan periplasmic binding protein from the Tripartite Tricarboxylate Transporter (TTT) family in Rhodopseudomonas palustris Leonardo T. Rosa , Samuel R. Dix , John B. Rafferty , David J. Kelly The Febs Journal DOI: 10.1111/febs.14304 Diamond Proposal Number(s): [12788] Show Abstract ▼ Abstract: The Tripartite Tricarboxylate Transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate binding-protein (SBP) for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP-ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with KD values in the μM range. The highest affinity was found for the C6-dicarboxylate adipate (1,6-hexanedioate). Crystal structures of AdpC with either adipate or 2-oxoadipate bound revealed a lack of positively charged amino-acids in the binding pocket and showed that water molecules are involved in bridging hydrogen bonds to the substrate, a conserved feature in the TTT SBP family that is distinct from other types of SBP. In AdpC, both of the ligand carboxylate groups and a linear chain conformation are needed for coordination in the binding pocket. RT-PCR showed that adpC expression is upregulated by low environmental adipate concentrations, suggesting adipate is a physiologically relevant substrate but as adpC is not genetically linked to any TTT membrane transport genes, the role of AdpC may be in signalling rather than transport. Our data expands the known ligands for TTT systems and identifies a novel high-affinity binding-protein for adipate, an important industrial chemical intermediate and food additive.	Oct 2017
I03-Macromolecular Crystallography	Structural insights into dynamics of RecU–HJ complex formation elucidates key role of NTR and stalk region toward formation of reactive state Sagar Khavnekar , Sarath Chandra Dantu , Svetlana Sedelnikova , Sylvia Ayora , John Rafferty , Avinash Kale Nucleic Acids Research DOI: 10.1093/nar/gkw1165 Diamond Proposal Number(s): [300] Open Access Show Abstract ▼ Abstract: Holliday junction (HJ) resolving enzyme RecU is involved in DNA repair and recombination. We have determined the crystal structure of inactive mutant (D88N) of RecU from Bacillus subtilis in complex with a 12 base palindromic DNA fragment at a resolution of 3.2 Å. This structure shows the stalk region and the essential N-terminal region (NTR) previously unseen in our DNA unbound structure. The flexible nature of the NTR in solution was confirmed using SAXS. Thermofluor studies performed to assess the stability of RecU in complex with the arms of an HJ indicate that it confers stability. Further, we performed molecular dynamics (MD) simulations of wild type and an NTR deletion variant of RecU, with and without HJ. The NTR is observed to be highly flexible in simulations of the unbound RecU, in agreement with SAXS observations. These simulations revealed domain dynamics of RecU and their role in the formation of complex with HJ. The MD simulations also elucidate key roles of the NTR, stalk region, and breathing motion of RecU in the formation of the reactive state.	Nov 2016
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography Data acquisition Diagnostics Health Physics	Direct observation of DNA threading in flap endonuclease complexes Faizah Almalki , Claudia Flemming , Jing Zhang , Min Feng , Svetlana E Sedelnikova , Tom Ceska , J B Rafferty , Jon Sayers , P J Artymiuk Nature Structural & Molecular Biology 23, 640 - 646 DOI: 10.1038/nsmb.3241 Show Abstract ▼ Abstract: Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism.	Jun 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species Chatchawal Phansopa , Sumita Roy , J B Rafferty , C. W. ian Douglas , Jagroop Pandhal , Phillip C. Wright , David J Kelly , Graham p. Stafford Biochemical Journal 458, 499 - 511 DOI: 10.1042/BJ20131415 PMID: 24351045 Diamond Proposal Number(s): [8987, 7423] Open Access Show Abstract ▼ Abstract: Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), WHERE NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).	Mar 2014
I02-Macromolecular Crystallography	Mutants of phage bIL67 RuvC with enhanced Holliday junction binding selectivity and resolution symmetry Victoria Green , Fiona A. Curtis , Svetlana Sedelnikova , John B. Rafferty , Gary J. Sharples Molecular Microbiology DOI: 10.1111/mmi.12343 PMID: 23888987 Open Access Show Abstract ▼ Abstract: Viral and bacterial Holliday junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively‐charged residues in the outer sides of the junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild‐type, while retaining the ability to bind X junctions. Crucially, these two variants cleaved Holliday junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.	Jul 2013
I02-Macromolecular Crystallography	The CouPSTU and TarPQM Transporters in Rhodopseudomonas palustris: Redundant, Promiscuous Uptake Systems for Lignin-Derived Aromatic Substrates Robert C. Salmon , Matthew J. Cliff , J B Rafferty , David J. Kelly , Vladimir N. Uversky PLoS ONE 8 (3) DOI: 10.1371/journal.pone.0059844 PMID: 23555803 Open Access Show Abstract ▼ Abstract: The biodegradation of lignin, one of the most abundant carbon compounds on Earth, has important biotechnological applications in the derivation of useful products from lignocellulosic wastes. The purple photosynthetic bacterium Rhodopseudomonas palustris is able to grow photoheterotrophically under anaerobic conditions on a range of phenylpropeneoid lignin monomers, including coumarate, ferulate, caffeate, and cinnamate. RPA1789 (CouP) is the periplasmic binding-protein component of an ABC system (CouPSTU; RPA1789, RPA1791–1793), which has previously been implicated in the active transport of this class of aromatic substrate. Here, we show using both intrinsic tryptophan fluorescence and isothermal titration calorimetry that CouP binds a range of phenylpropeneoid ligands with Kd values in the nanomolar range. The crystal structure of CouP with ferulate as the bound ligand shows H-bond interactions between the 4-OH group of the aromatic ring with His309 and Gln305. H-bonds are also made between the carboxyl group on the ferulate side chain and Arg197, Ser222, and Thr102. An additional transport system (TarPQM; RPA1782–1784), a member of the tripartite ATP-independent periplasmic (TRAP) transporter family, is encoded at the same locus as rpa1789 and several other genes involved in coumarate metabolism. We show that the periplasmic binding-protein of this system (TarP; RPA1782) also binds coumarate, ferulate, caffeate, and cinnamate with nanomolar Kd values. Thus, we conclude that R. palustris uses two redundant but energetically distinct primary and secondary transporters that both employ high-affinity periplasmic binding-proteins to maximise the uptake of lignin-derived aromatic substrates from the environment. Our data provide a detailed thermodynamic and structural basis for understanding the interaction of lignin-derived aromatic substrates with proteins and will be of use in the further exploitation of the flexible metabolism of R. palustris for anaerobic aromatic biotransformations.	Mar 2013
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase J. P. Afonso , K. Chintakayala , Chatrudee Suwannachart , Svetlana E. Sedelnikova , K. Giles , J. B. Hoyes , P. Soultanas , J B Rafferty , N. J. Oldham Nucleic Acids Research 41 (9), 5115 - 5126 DOI: 10.1093/nar/gkt173 PMID: 23525462 Diamond Proposal Number(s): [7423, 1218, 300] Open Access Show Abstract ▼ Abstract: The clamp-loader complex plays a crucial role in DNA replication by loading the b-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric q3-d-d0 Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli.	Mar 2013

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