I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 19281]
Open Access
Abstract: Macrocyclization and multiple backbone N-methylations can significantly improve the pharmacological properties of peptides. Since chemical synthesis of such compounds is often challenging, enzyme-based production platforms are an interesting option. Here, we characterized OphP, a serine peptidase involved in the cyclization of omphalotins, a group of ribosomally produced dodecapeptides with multiple backbone N-methylations. OphP displays robust peptidase and macrocyclase activity towards multiply α-N-methylated peptides of various lengths and composition derived from the omphalotin precursor protein OphMA. In addition, OphP processes, with lower efficiency, peptides unrelated to OphMA, containing a MeGly, MeAla or Pro residue at the P1 site. Structural analysis reveals that OphP adopts a canonical prolyl oligopeptidase fold but, unlike other enzymes of this enzyme family, recognizes its substrates by their hydrophobic and multiply backbone N-methylated core rather than by the follower peptide. The activity of OphP could be harnessed for the enzymatic production of therapeutic peptides.
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Jul 2025
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Abner
Velazco
,
Thomas
Glen
,
Sven
Klumpe
,
Avery
Pennington
,
Jianguo
Zhang
,
Jake L. R.
Smith
,
Calina
Glynn
,
William
Bowles
,
Maryna
Kobylynska
,
Roland A.
Fleck
,
James H.
Naismith
,
Judy S.
Kim
,
Michele C.
Darrow
,
Michael
Grange
,
Angus I.
Kirkland
,
Maud
Dumoux
Open Access
Abstract: Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in a representation of their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional model of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the use of serial FIB/SEM imaging-based methods is often limited due to charging artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to attenuate these artefacts, allowing the observation of biological features that otherwise would be masked or distorted. We apply our method to samples where inherent features were not visible using conventional scanning. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E. gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. The proposed alternative scanning method could also be applied to imaging other non-conductive specimens in SEM.
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Jun 2025
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I03-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
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Audrey
Le Bas
,
Bradley R.
Clarke
,
Tanisha
Teelucksingh
,
Micah
Lee
,
Kamel
El Omari
,
Andrew M.
Giltrap
,
Stephen A.
Mcmahon
,
Hui
Liu
,
John H.
Beale
,
Vitaliy
Mykhaylyk
,
Ramona
Duman
,
Neil G.
Paterson
,
Philip N.
Ward
,
Peter J.
Harrison
,
Miriam
Weckener
,
Els
Pardon
,
Jan
Steyaert
,
Huanting
Liu
,
Andrew
Quigley
,
Benjamin G.
Davis
,
Armin
Wagner
,
Chris
Whitfield
,
James H.
Naismith
Diamond Proposal Number(s):
[33941]
Open Access
Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.
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Jan 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Abstract: Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-D-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70-80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80 °C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)8-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.
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Dec 2024
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Open Access
Abstract: Background:
A key issue with the established method of cryo-electron tomography (Cryo- ET) often lies in the challenge of accurately locating complexes or proteins of interest within the crowded cellular environment of the generated tomograms. This project aims to use cryo-scanning transmission electron microscopy (STEM) to highlight regions of tomograms containing the protein of interest by tagging with a minimally-sized heavy atom nanoparticle for downstream processing. A fine balance must be achieved between resolution, signal-to-noise ratio (SNR), depth of focus, and damage to the lamella.
Methods:
A scan generator offers alternative (non-raster) methods of scanning the beam to offset damage and allow higher electron fluences to be used without compromising ice quality. Small raster and interleaved scans (< 200 nm) were compared in the same quantifoil hole of vitreous ice, using an equal overall electron fluence and flux.
Analysis of elastic scattering cross section theory yields an approximate minimal size of nanoparticles for detection in STEM of vitreous amorphous specimens. Single particle-like sample preparation was employed to freeze varying sizes of gold nanoparticles (0.8-4 nm) in different thicknesses of ice. These were subsequently exposed to STEM to assess a drop-off in SNR with increasing collection angles using an annular dark field detector.
Results:
Scanning in an alternative fashion using long dwell times demonstrated a significant reduction in mass loss. Raster scanning appeared to be marginally better than interleaved scanning when using shorter dwell times (20 μs), as evidenced by greater loss of intensity in the scanned areas, normalised to reference areas taken within the same hole of vitreous ice. However, using longer dwell times (250 μs or 500 μs) reversed this effect, showing raster scanning to be significantly more damaging than interleaved, melting the ice completely in thin samples for raster scanning, whilst maintaining the ice intact using an interleaved sequence.
Conclusions:
These findings provide valuable first steps toward optimizing cryo-STEM imaging for detecting nanoparticles and correlating these findings with in-situ Transmission Electron Microscopy (TEM).
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Oct 2024
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I04-Macromolecular Crystallography
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Lachlan P.
Deimel
,
Lucile
Moynie
,
Guoxuan
Sun
,
Viliyana
Lewis
,
Abigail
Turner
,
Charles J.
Buchanan
,
Sean A.
Burnap
,
Mikhail
Kutuzov
,
Carolin M.
Kobras
,
Yana
Demyaneko
,
Shabaz
Mohammed
,
Mathew
Stracy
,
Weston B.
Struwe
,
Andrew J.
Baldwin
,
James
Naismith
,
Benjamin G.
Davis
,
Quentin J.
Sattentau
Open Access
Abstract: Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses.
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Aug 2024
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Open Access
Abstract: For cryo-electron tomography (cryo-ET) of beam-sensitive biological specimens, a planar sample geometry is typically used. As the sample is tilted, the effective thickness of the sample along the direction of the electron beam increases and the signal-to-noise ratio concomitantly decreases, limiting the transfer of information at high tilt angles. In addition, the tilt range where data can be collected is limited by a combination of various sample-environment constraints, including the limited space in the objective lens pole piece and the possible use of fixed conductive braids to cool the specimen. Consequently, most tilt series are limited to a maximum of ±70°, leading to the presence of a missing wedge in Fourier space. The acquisition of cryo-ET data without a missing wedge, for example using a cylindrical sample geometry, is hence attractive for volumetric analysis of low-symmetry structures such as organelles or vesicles, lysis events, pore formation or filaments for which the missing information cannot be compensated by averaging techniques. Irrespective of the geometry, electron-beam damage to the specimen is an issue and the first images acquired will transfer more high-resolution information than those acquired last. There is also an inherent trade-off between higher sampling in Fourier space and avoiding beam damage to the sample. Finally, the necessity of using a sufficient electron fluence to align the tilt images means that this fluence needs to be fractionated across a small number of images; therefore, the order of data acquisition is also a factor to consider. Here, an n-helix tilt scheme is described and simulated which uses overlapping and interleaved tilt series to maximize the use of a pillar geometry, allowing the entire pillar volume to be reconstructed as a single unit. Three related tilt schemes are also evaluated that extend the continuous and classic dose-symmetric tilt schemes for cryo-ET to pillar samples to enable the collection of isotropic information across all spatial frequencies. A fourfold dose-symmetric scheme is proposed which provides a practical compromise between uniform information transfer and complexity of data acquisition.
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Jun 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Katy
Cornish
,
Jiandong
Huo
,
Luke
Jones
,
Parul
Sharma
,
Joseph W.
Thrush
,
Sahar
Abdelkarim
,
Anja
Kipar
,
Siva
Ramadurai
,
Miriam
Weckener
,
Halina
Mikolajek
,
Sai
Liu
,
Imogen
Buckle
,
Eleanor
Bentley
,
Adam
Kirby
,
Ximeng
Han
,
Stephen M.
Laidlaw
,
Michelle
Hill
,
Lauren
Eyssen
,
Chelsea
Norman
,
Audrey
Le Bas
,
John
Clarke
,
William
James
,
James P.
Stewart
,
Miles
Carroll
,
James
Naismith
,
Raymond J.
Owens
Diamond Proposal Number(s):
[27031]
Open Access
Abstract: The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics.
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Jun 2024
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B23-Circular Dichroism
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Cédric
Couturier
,
Quentin
Ronzon
,
Giulia
Lattanzi
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Iain
Lingard
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Sebastien
Coyne
,
Veronique
Cazals
,
Nelly
Dubarry
,
Stephane
Yvon
,
Corinne
Leroi-Geissler
,
Obdulia Rabal
Gracia
,
Joanne
Teague
,
Sylvie
Sordello
,
David
Corbett
,
Caroline
Bauch
,
Chantal
Monlong
,
Lloyd
Payne
,
Thomas
Taillier
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Hazel
Fuchs
,
Mark
Broenstrup
,
Peter H.
Harrison
,
Lucile
Moynie
,
Abirami
Lakshminarayanan
,
Tiberiu-Marius
Gianga
,
Rohanah
Hussain
,
James H.
Naismith
,
Michael
Mourez
,
Eric
Bacqué
,
Fredrik
Björkling
,
Jean-Francois
Sabuco
,
Henrik
Franzyk
Diamond Proposal Number(s):
[26447]
Abstract: Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.
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Feb 2024
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Open Access
Abstract: Simulations of cryo-electron microscopy (cryo-EM) images of biological samples can be used to produce test datasets to support the development of instrumentation, methods, and software, as well as to assess data acquisition and analysis strategies. To be useful, these simulations need to be based on physically realistic models which include large volumes of amorphous ice. The gold standard model for EM image simulation is a physical atom-based ice model produced using molecular dynamics simulations. Although practical for small sample volumes; for simulation of cryo-EM data from large sample volumes, this can be too computationally expensive. We have evaluated a Gaussian Random Field (GRF) ice model which is shown to be more computationally efficient for large sample volumes. The simulated EM images are compared with the gold standard atom-based ice model approach and shown to be directly comparable. Comparison with experimentally acquired data shows the Gaussian random field ice model produces realistic simulations. The software required has been implemented in the Parakeet software package and the underlying atomic models are available online for use by the wider community.
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Nov 2023
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