I23-Long wavelength MX
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Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. CCHFV is shortlisted as a priority pathogen by the World Health Organization. It is a (-)ssRNA virus of the family Nairoviridae in the Bunyavirales order. The CCHFV membrane fusion glycoprotein Gc has been shown to be the main target of the host neutralizing antibody response, whereas a secreted glycoprotein GP38 was demonstrated to be the target of non-neutralizing protective antibodies. However, structural information on the viral glycoproteins as well as antibody-mediated neutralization mechanisms has been lacking for members of the nairovirus family. The first part of this thesis describes the structure of monomeric prefusion CCHFV Gc bound to the antigen binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show that the two Fabs act in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The above structures also revealed the neutralization mechanism of antibodies targeting 6 different antigenic sites on CCHFV Gc, a few of which were further validated with structural studies. Describing CCHFV neutralization at the mechanistic level, our data guide the design of future therapeutic antibodies and will likewise support the design of protective CCHFV vaccines. The second part of this thesis presents the structure of GP38, which reveals a novel protein fold. This study also describes the structure of a mouse antibody 13G8 bound to GP38; this antibody has been shown to protect against CCHFV. Mapping of the epitopes of several human GP38 antibodies onto the GP38 structure shows competition with the 13G8 epitope and identification of additional antigenic sites. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. Collectively both these studies provide the molecular underpinnings essential for developing CCHFV-specific medical countermeasures for epidemic preparedness.
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Apr 2022
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I04-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
Krios III-Titan Krios III at Diamond
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Paola
Lanzoni-Mangutchi
,
Oishik
Banerji
,
Jason
Wilson
,
Anna
Barwinska-Sendra
,
Joseph A.
Kirk
,
Filipa
Vaz
,
Shauna
O’beirne
,
Arnaud
Basle
,
Kamel
El Omari
,
Armin
Wagner
,
Neil F.
Fairweather
,
Gillian R.
Douce
,
Per A.
Bullough
,
Robert P.
Fagan
,
Paula
Salgado
Diamond Proposal Number(s):
[15523, 18598, 19832]
Open Access
Abstract: Many bacteria and archaea possess a two-dimensional protein array, or S-layer, that covers the cell surface and plays crucial roles in cell physiology. Here, we report the crystal structure of SlpA, the main S-layer protein of the bacterial pathogen Clostridioides difficile, and use electron microscopy to study S-layer organisation and assembly. The SlpA crystal lattice mimics S-layer assembly in the cell, through tiling of triangular prisms above the cell wall, interlocked by distinct ridges facing the environment. Strikingly, the array is very compact, with pores of only ~10 Å in diameter, compared to other S-layers (30–100 Å). The surface-exposed flexible ridges are partially dispensable for overall structure and assembly, although a mutant lacking this region becomes susceptible to lysozyme, an important molecule in host defence. Thus, our work gives insights into S-layer organisation and provides a basis for development of C. difficile-specific therapeutics.
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Feb 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I23-Long wavelength MX
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Leandro
Oliveira Bortot
,
Victor
Lopes Rangel
,
Francesca A.
Pavlovici
,
Kamel
El Omari
,
Armin
Wagner
,
Jose
Brandao-Neto
,
Romain
Talon
,
Frank
Von Delft
,
Andrew G.
Reidenbach
,
Sonia M.
Vallabh
,
Eric
Vallabh Minikel
,
Stuart
Schreiber
,
Maria Cristina
Nonato
Diamond Proposal Number(s):
[18954]
Abstract: Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.
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Dec 2021
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I23-Long wavelength MX
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Matthew
Herdman
,
Andriko
Von Kugelgen
,
Danguole
Kureisaite-Ciziene
,
Ramona
Duman
,
Kamel
El Omari
,
Elspeth F.
Garman
,
Andreas
Kjaer
,
Dimitrios
Kolokouris
,
Jan
Lowe
,
Armin
Wagner
,
Phillip J.
Stansfeld
,
Tanmay A. M.
Bharat
Open Access
Abstract: Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.
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Nov 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I23-Long wavelength MX
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Abstract: Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an "undruggable" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors.
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Aug 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[18565]
Abstract: Some bacteria can survive in the absence of oxygen by using a metal, such as iron, instead. To do this, they require a system for getting electrons from the inside of the cell to the outside, which is usually prevented by the insulating membrane surrounding the cell. There was no known system that could do this.
Previous research identified three genes in the aquatic bacterium Shewanella oneidensis that could move electrons out of the cell. However, it was unclear how these three genes could cause such an effect. Researchers from the University of East Anglia investigated how the products of these genes could assemble into something allowing electron transport across the outer membrane of the cell.
They collected X-ray data on Macromolecular Crystallography (MX) beamlines I03 and I04, using intense, tuneable X-rays to collect data from the small and fragile protein crystals. Using this data, the team were able to identify the positions of the 20 iron atoms inside the protein structure, which allowed the building of the complete structure. For the first time, they were able to see how nature assembles wires out of biological material, and how to build complexes to allow electrons to flow into and out of a living cell. Complexes such as this have a range of potential applications, including biomining using microbes to extract copper and gold from low-grade ores.
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Jul 2021
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I23-Long wavelength MX
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Abstract: The flow of potassium ions across the cell membrane is regulated by K2P potassium channels, controlling electrical activity in the brain, heart and nervous system. These channels have important roles in pain, migraine, depression, anaesthetic responses, arrhythmias, hypertension and lung injury responses. K channels regulate ion flow using a selectivity filter (C-type) gate. However, previous
2P
structural studies have not captured the gating mechanisms, and they remain poorly understood. Currently, there are no drugs available
that selectively target K2P potassium channels. To develop new ways to control their function, we need to understand more about the gating mechanisms and their roles in human biology.
In work published in Science Advances, researchers from Diamond Light Source, University of California, San Francisco and the University Pittsburgh, USA, combined X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics and electrophysiology to uncover the changes that occur in K2P C-type gating. At Diamond, the team used the Long-Wavelength Macromolecular Crystallography (MX) beamline (I23) to observe changes in potassium ions within the channel directly. I23 is the only beamline in the world optimised to measure signals from potassium ions directly, and its unique capabilities were central to these studies. The results show that K2P gating involves pinching and dilation of two key elements of the channel selectivity filter gate. These structural changes are accompanied by the loss of potassium ions. Including a small molecule activator (ML335) suppressed these changes and demonstrated how stabilisation of the selectivity filter gate facilitates ion flow through the channel.
These studies show that small molecule activators bind to and stabilise the K2P selectivity filter gate, preventing the pinching and dilation conformational changes and loss of potassium ions that lead to channel inactivation. These findings open a path to develop new K2P channel- directed drugs to treat pain, ischemia (restricted blood flow), depression and lung injury.
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Jul 2021
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I04-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[20145]
Open Access
Abstract: Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA–protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.
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Jul 2021
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I04-Macromolecular Crystallography
I23-Long wavelength MX
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Open Access
Abstract: During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.
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May 2021
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I23-Long wavelength MX
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Open Access
Abstract: In this paper a practical solution for the reconstruction and segmentation of low-contrast X-ray tomographic data of protein crystals from the long-wavelength macromolecular crystallography beamline I23 at Diamond Light Source is provided. The resulting segmented data will provide the path lengths through both diffracting and non-diffracting materials as basis for analytical absorption corrections for X-ray diffraction data taken in the same sample environment ahead of the tomography experiment. X-ray tomography data from protein crystals can be difficult to analyse due to very low or absent contrast between the different materials: the crystal, the sample holder and the surrounding mother liquor. The proposed data processing pipeline consists of two major sequential operations: model-based iterative reconstruction to improve contrast and minimize the influence of noise and artefacts, followed by segmentation. The segmentation aims to partition the reconstructed data into four phases: the crystal, mother liquor, loop and vacuum. In this study three different semi-automated segmentation methods are experimented with by using Gaussian mixture models, geodesic distance thresholding and a novel morphological method, RegionGrow, implemented specifically for the task. The complete reconstruction-segmentation pipeline is integrated into the MPI-based data analysis and reconstruction framework Savu, which is used to reduce computation time through parallelization across a computing cluster and makes the developed methods easily accessible.
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May 2021
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