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32 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	Mutations in the N-domain of aryl hydrocarbon receptor interacting protein affect interactions with heat shock protein 90β and phosphodiesterase 4A5 Marita Vella , Iain W. Manfield , Brandon C. Seychell , Chi H. Trinh , Robert Rambo , G. Nasir Khan , Josanne Vassallo , Therese Hunter , Gary J. Hunter Biochimie 272 DOI: 10.1016/j.biochi.2024.09.005 Diamond Proposal Number(s): [15574, 18536] Open Access Show Abstract ▼ Abstract: The aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation involving the aryl hydrocarbon receptor. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. Full length AIP harbouring N-domain mutations (R9Q, R16H, V49M and K103R) were purified from E.coli utilizing a methodology that maintained structural integrity and monomeric stability. Mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. The mutations studied lowered the binding affinity of AIP towards two of its binding partners; heat shock protein 90β and phosphodiesterase 4A5 (PDE4A5). The inhibition of phosphodiesterase activity by AIP was also greatly reduced by all mutants. While previously published data has mainly concentrated on the tetratricopeptide repeats of the C-domain of AIP, we present clear evidence that AIP N-domain mutations play a significant role in two protein:protein interactions with partner proteins. The complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways.	Sep 2024
B21-High Throughput SAXS	Using atomistic solution scattering modelling to elucidate the role of the Fc glycans in human IgG4 Valentina A. Spiteri , James Doutch , Robert P. Rambo , Jayesh S. Bhatt , Jayesh Gor , Paul A. Dalby , Stephen J. Perkins Plos One 19 DOI: 10.1371/journal.pone.0300964 Diamond Proposal Number(s): [18022] Open Access Show Abstract ▼ Abstract: Human immunoglobulin G (IgG) exists as four subclasses IgG1-4, each of which has two Fab subunits joined by two hinges to a Fc subunit. IgG4 has the shortest hinge with 12 residues. The Fc subunit has two glycan chains, but the importance of glycosylation is not fully understood in IgG4. Here, to evaluate the stability and structure of non-glycosylated IgG4, we performed a multidisciplinary structural study of glycosylated and deglycosylated human IgG4 A33 for comparison with our similar study of human IgG1 A33. After deglycosylation, IgG4 was found to be monomeric by analytical ultracentrifugation; its sedimentation coefficient of 6.52 S was reduced by 0.27 S in reflection of its lower mass. X-ray and neutron solution scattering showed that the overall Guinier radius of gyration RG and its cross-sectional values after deglycosylation were almost unchanged. In the P(r) distance distribution curves, the two M1 and M2 peaks that monitor the two most common distances within IgG4 were unchanged following deglycosylation. Further insight from Monte Carlo simulations for glycosylated and deglycosylated IgG4 came from 111,382 and 117,135 possible structures respectively. Their comparison to the X-ray and neutron scattering curves identified several hundred best-fit models for both forms of IgG4. Principal component analyses showed that glycosylated and deglycosylated IgG4 exhibited different conformations from each other. Within the constraint of unchanged RG and M1-M2 values, the glycosylated IgG4 models showed more restricted Fc conformations compared to deglycosylated IgG4, but no other changes. Kratky plots supported this interpretation of greater disorder upon deglycosylation, also observed in IgG1. Overall, these more variable Fc conformations may demonstrate a generalisable impact of deglycosylation on Fc structures, but with no large conformational changes in IgG4 unlike those seen in IgG1.	Apr 2024
	An innovative update and expansion programme to the UK’s national synchrotron Gianluigi A. Botton , Peter Coll , Andrew J. Dent , Sarnjeet Dhesi , Sky French , David R. Hall , Joseph Hriljac , Sarah Macdonell , Sofia Diaz-Moreno , Chris Nicklin , Paul Quinn , Robert Rambo , Kawal Sawhney , Richard P. Walker , Martin A. Walsh , Molly Pekarik-Fry Synchrotron Radiation News DOI: 10.1080/08940886.2024.2312066 Open Access Show Abstract ▼ Abstract: A look at the Diamond-II upgrade programme	Mar 2024
B21-High Throughput SAXS	The SKMT Algorithm: A method for assessing and comparing underlying protein entanglement Arron Bale , Robert Rambo , Christopher Prior Plos Computational Biology 19 DOI: 10.1371/journal.pcbi.1011248 Open Access Show Abstract ▼ Abstract: We present fast and simple-to-implement measures of the entanglement of protein tertiary structures which are appropriate for highly flexible structure comparison. These are performed using the SKMT algorithm, a novel method of smoothing the Cα backbone to achieve a minimal complexity curve representation of the manner in which the protein’s secondary structure elements fold to form its tertiary structure. Its subsequent complexity is characterised using measures based on the writhe and crossing number quantities heavily utilised in DNA topology studies, and which have shown promising results when applied to proteins recently. The SKMT smoothing is used to derive empirical bounds on a protein’s entanglement relative to its number of secondary structure elements. We show that large scale helical geometries dominantly account for the maximum growth in entanglement of protein monomers, and further that this large scale helical geometry is present in a large array of proteins, consistent across a number of different protein structure types and sequences. We also show how these bounds can be used to constrain the search space of protein structure prediction from small angle x-ray scattering experiments, a method highly suited to determining the likely structure of proteins in solution where crystal structure or machine learning based predictions often fail to match experimental data. Finally we develop a structural comparison metric based on the SKMT smoothing which is used in one specific case to demonstrate significant structural similarity between Rossmann fold and TIM Barrel proteins, a link which is potentially significant as attempts to engineer the latter have in the past produced the former. We provide the SWRITHE interactive python notebook to calculate these metrics.	Nov 2023
B21-High Throughput SAXS I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Crystal structure and solution state of the C-terminal head region of the narmovirus receptor binding protein Alice J. Stelfox , Kasopefoluwa Y. Oguntuyo , Ilona Rissanen , Karl Harlos , Robert Rambo , Benhur Lee , Thomas Bowden Mbio DOI: 10.1128/mbio.01391-23 Diamond Proposal Number(s): [10627, 14744] Open Access Show Abstract ▼ Abstract: Increased viral surveillance has led to the isolation and identification of numerous uncharacterized paramyxoviruses, rapidly expanding our understanding of paramyxoviral diversity beyond the bounds of known genera. Despite this diversity, a key feature that unites paramyxoviruses is the presence of a receptor-binding protein (RBP), which facilitates host-cell attachment and plays a fundamental role in determining host range. Here, we study the RBP presented on the surface of rodent-borne paramyxoviruses Mossman and Nariva (MosV and NarV, respectively), viruses that constitute founding members of the recently defined Narmovirus genus within the Paramyxoviridae family. Crystallographic analysis of the C-terminal head region of the dimeric MosV and NarV RBPs demonstrates that while these glycoproteins retain the canonical six-bladed β-propeller fold found in other paramyxoviral RBPs, they lack the structural motifs associated with established paramyxovirus host-cell receptor entry pathways. Consistent with MosV-RBP and NarV-RBP undergoing a distinct entry pathway from other characterized paramyxoviruses, structure-based phylogenetic analysis demonstrates that these six-bladed β-propeller head domains form a singular structural class that is distinct from other paramyxoviral RBPs. Additionally, using an integrated crystallographic and small-angle X-ray scattering analysis, we confirm that MosV-RBP and NarV-RBP form homodimeric arrangements that are distinct from those adopted by other paramyxovirus RBPs. Altogether, this investigation provides a molecular-level blueprint of the narmovirus RBP that broadens our understanding of the structural space and functional diversity available to paramyxovirus RBPs.	Sep 2023
B21-High Throughput SAXS	Biophysical characterization of the structure of a SARS-CoV-2 self-amplifying RNA (saRNA) vaccine Daniel Myatt , Lewis Wharram , Charlotte Graham , John Liddell , Harvey Branton , Claire Pizzey , Nathan P. Cowieson , Robert Rambo , Robin J. Shattock Biology Methods And Protocols 8 DOI: 10.1093/biomethods/bpad001 Open Access Show Abstract ▼ Abstract: The current SARS-Covid-2 (SARS-CoV-2) pandemic has led to an acceleration of messenger ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA), has required concomitant development of analytical characterization techniques. Characterizing the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This article describes the biophysical characterization of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterize the IMP-1 RNA molecule. In this article, we use ultraviolet spectroscopy, dynamic light scattering, size-exclusion chromatography small-angle X-ray scattering and circular dichroism to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule.	Jan 2023
B21-High Throughput SAXS I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Primary and secondary functions of HLA-E are determined by stability and conformation of the peptide-bound complexes Lucy C. Walters , Daniel Rozbesky , Karl Harlos , Max Quastel , Hong Sun , Sebastian Springer , Robert P. Rambo , Fiyaz Mohammed , E. Yvonne Jones , Andrew J. Mcmichael , Geraldine M. Gillespie Cell Reports 39 DOI: 10.1016/j.celrep.2022.110959 Diamond Proposal Number(s): [19946] Open Access Show Abstract ▼ Abstract: MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8+ T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination.	Jun 2022
B21-High Throughput SAXS	Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses Valentina A. Spiteri , Margaret Goodall , James Doutch , Robert P. Rambo , Jayesh Gor , Stephen J. Perkins Journal Of Biological Chemistry 40 DOI: 10.1016/j.jbc.2021.100995 Open Access Show Abstract ▼ Abstract: Human immunoglobulin IgG3 possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved glycosylation sites in the Fc region is unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82-6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) of both forms of IgG3 showed a maximum length of 25-28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modelling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically-realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each of the two forms of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively-restricted Fab and Fc conformations joined by an extended semi-rigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2 and IgG4.	Jul 2021
B16-Test Beamline B21-High Throughput SAXS	Structural basis for the coiled-coil architecture of human CtIP C. R. Morton , N. J. Rzechorzek , J. D. Maman , M. Kuramochi , H. Sekiguchi , R. Rambo , Y. C. Sasaki , O. R. Davies , Luca Pellegrini Open Biology 11 DOI: 10.1098/rsob.210060 Open Access Show Abstract ▼ Abstract: The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.	Jun 2021
B21-High Throughput SAXS	Solution structure of deglycosylated human IgG1 shows the role of CH2 glycans in its conformation Valentina A. Spiteri , James Doutch , Robert P. Rambo , Jayesh Gor , Paul A. Dalby , Stephen J. Perkins Biophysical Journal DOI: 10.1016/j.bpj.2021.02.038 Diamond Proposal Number(s): [18022] Show Abstract ▼ Abstract: The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is comprised of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG, however their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. Following deglycosylation using PNGase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16-0.27 S. This change was attributed to the reduction in mass following glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. While the radius of gyration RG was unchanged, the cross-sectional radius of gyration, RXS-1, increased by 0.1 nm and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed following deglycosylation. To explain these changes, atomistic scattering modelling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 X-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, while the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the FcγR receptor in deglycosylated IgG1.	Mar 2021

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