VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[28534, 34263]
Open Access
Abstract: Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme–nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.
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Feb 2026
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Muhammad
Saleem
,
Chan
Li
,
Bubacarr G.
Kaira
,
Alexander K.
Brown
,
Monika
Pathak
,
Shabir
Najmudin
,
Nathan
Cowieson
,
Ingrid
Dreveny
,
Clare
Wilson
,
Aleksandr
Shamanaev
,
David
Gailani
,
Stephanie A.
Smith
,
James H.
Morrissey
,
Helen
Philippou
,
Jonas
Emsley
Diamond Proposal Number(s):
[37038]
Open Access
Abstract: Factor XIIa (FXIIa) is generated from its zymogen factor XII (FXII) by contact with polyanions such as inorganic polyphosphates. FXIIa cleaves the substrates prekallikrein and factor XI, triggering inflammatory cascades and plasma coagulation. From the N-terminus, FXII has fibronectin type II (FnII), epidermal growth factor-1 (EGF1), fibronectin type I (FnI), EGF2 and kringle domains. The N-terminal domains of FXII mediate polyanion and Zn2+ binding. To understand how ligand binding to polyanions and Zn2+ is coordinated across multiple domains, we determined the crystal structure of recombinant FXII domains 1–5 (FXIIHC5) to 3.4 Å resolution. A separate crystal structure of the isolated FXII FnII domain at 1.2 Å resolution revealed two bound Zn2+ ions. In FXIIHC5 a head-to-tail interaction is formed between the FnII and kringle domains, co-localizing the lysine-binding sites of the kringle domain and the cation-binding site of the FnII domain. Two FXIIHC5 monomers interlock, burying a large surface area of 2067 Å2, such that two kringle domains point outwards separated by a distance of 20 Å. The polyanion-binding site in the EGF1 domain is localized onto a plane together with the FnII and FnI domains. Using native mass spectrometry, we detected a major FXIIHC5 monomer peak and a minor dimer peak. Small-angle X-ray scattering and gel-filtration chromatography revealed the presence of monomers and dimers in solution. These FXII N-terminal domain structures provide a holistic framework to understand how the mosaic domain structure of FXII assembles diverse ligand-binding sites in three dimensions.
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Jul 2025
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I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: Multi-crystal processing of X-ray diffraction data has become highly automated to keep pace with the current high-throughput capabilities afforded by beamlines. A significant challenge, however, is the automated clustering of such data based on subtle differences such as ligand binding or conformational shifts. Intensity-based hierarchical clustering has been shown to be a viable method of identifying such subtle structural differences, but the interpretation of the resulting dendrograms is difficult to automate. Using isomorphous crystals of bovine, porcine and human insulin, the existing clustering methods in the multi-crystal processing software xia2.multiplex were validated and their limits were tested. It was determined that weighting the pairwise correlation coefficient calculations with the intensity uncertainties was required for accurate calculation of the pairwise correlation coefficient matrix (correlation clustering) and dimension optimization was required when expressing this matrix as a set of coordinates representing data sets (cosine-angle clustering). Finally, the introduction of the OPTICS spatial density-based clustering algorithm into DIALS allowed the automatic output of species-pure clusters of bovine, porcine and human insulin data sets.
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Jun 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Suzan A. D.
Miranda
,
Lucas
Jahn
,
Elena V.
Blagova
,
Andrey A.
Lebedev
,
Dorotea R.
Segura
,
Mary A.
Stringer
,
Esben P.
Friis
,
Joao P. L.
Franco Cairo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Endo-galactosaminidases are an underexplored family of enzymes involved in the degradation of galactosaminogalactan (GAG) and other galactosamine-containing cationic exopolysaccharides produced by fungi and bacteria. These exopolysaccharides are part of the cell wall and extracellular matrix of microbial communities. Currently, these galactosaminidases are found in three distinct CAZy families: GH114, GH135 and GH166. Despite the widespread occurrence of these enzymes in nearly all bacterial and fungal clades, only limited biochemical and structural data are available for these three groups. To expand our knowledge of endo-galactosaminidases, we selected several sequences predicted to encode endo-galactosaminidases and produced them recombinantly for structural and functional studies. Only very few predicted proteins could be produced in soluble form, and activity against bacterial Pel (pellicle) polysaccharide could only be confirmed for one enzyme. Here, we report the structures of two bacterial and one fungal enzyme. Whereas the fungal enzyme belongs to family GH114, the two bacterial enzymes do not lie in the current GH families but instead define a new family, GH191. During structure solution we realized that crystals of all three enzymes had various defects including twinning and partial disorder, which in the case of a more severe pathology in one of the structures required the design of a specialized refinement/model-building protocol. Comparison of the structures revealed several features that might be responsible for the described activity pattern and substrate specificity compared with other GAG-degrading enzymes.
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May 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Alexandra
Males
,
Olga V.
Moroz
,
Elena
Blagova
,
Astrid
Munch
,
Gustav H.
Hansen
,
Annette H.
Johansen
,
Lars H.
Østergaard
,
Dorotea R.
Segura
,
Alexander
Eddenden
,
Anne V.
Due
,
Martin
Gudmand
,
Jesper
Salomon
,
Sebastian R.
Sørensen
,
Joao Paulo L.
Franco Cairo
,
Mark
Nitz
,
Roland A.
Pache
,
Rebecca M.
Vejborg
,
Sandeep
Bhosale
,
David J.
Vocadlo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Microorganisms are known to secrete copious amounts of extracellular polymeric substances (EPS) that form complex matrices around the cells to shield them against external stresses, to maintain structural integrity and to influence their environment. Many microorganisms also secrete enzymes that are capable of remodelling or degrading EPS in response to various environmental cues. One key enzyme class is the poly-β-1,6-linked N-acetyl-D-glucosamine (PNAG)-degrading glycoside hydrolases, of which the canonical member is dispersin B (DspB) from CAZy family GH20. We sought to test the hypothesis that PNAG-degrading enzymes would be present across family GH20, resulting in expansion of the sequence and structural space and thus the availability of PNAGases. Phylogenetic analysis revealed that several microorganisms contain potential DspB-like enzymes. Six of these were expressed and characterized, and four crystal structures were determined (two of which were in complex with the established GH20 inhibitor 6-acetamido-6-deoxy-castanospermine and one with a bespoke disaccharide β-1,6-linked thiazoline inhibitor). One enzyme expressed rather poorly, which restricted crystal screening and did not allow activity measurements. Using synthetic PNAG oligomers and MALDI-TOF analysis, two of the five enzymes tested showed preferential endo hydrolytic activity. Their sequences, having only 26% identity to the pioneer enzyme DspB, highlight the considerable array of previously unconsidered dispersins in nature, greatly expanding the range of potential dispersin backbones available for societal application and engineering.
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Mar 2025
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VMXi-Versatile Macromolecular Crystallography in situ
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Open Access
Abstract: A group of three deep-learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre-trained deep-learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug-discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets.
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Oct 2024
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I23-Long wavelength MX
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Open Access
Abstract: AlphaFold2 has revolutionized structural biology by offering unparalleled accuracy in predicting protein structures. Traditional methods for determining protein structures, such as X-ray crystallography and cryo-electron microscopy, are often time-consuming and resource-intensive. AlphaFold2 provides models that are valuable for molecular replacement, aiding in model building and docking into electron density or potential maps. However, despite its capabilities, models from AlphaFold2 do not consistently match the accuracy of experimentally determined structures, need to be validated experimentally and currently miss some crucial information, such as post-translational modifications, ligands and bound ions. In this paper, the advantages are explored of collecting X-ray anomalous data to identify chemical elements, such as metal ions, which are key to understanding certain structures and functions of proteins. This is achieved through methods such as calculating anomalous difference Fourier maps or refining the imaginary component of the anomalous scattering factor f′′. Anomalous data can serve as a valuable complement to the information provided by AlphaFold2 models and this is particularly significant in elucidating the roles of metal ions.
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Oct 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19951, 24732]
Open Access
Abstract: The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE–fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.
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Aug 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12579]
Open Access
Abstract: The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239–247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe–2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe–2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.
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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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