I22-Small angle scattering & Diffraction
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Tayyaba
Rabnawaz
,
Nathanael
Leung
,
Leonard C.
Nielsen
,
Robert A.
Harper
,
Richard M.
Shelton
,
Gabriel
Landini
,
Tim
Snow
,
Andy
Smith
,
Nick
Terrill
,
Marianne
Liebi
,
Tan
Sui
Diamond Proposal Number(s):
[20285]
Abstract: Dental caries, one of the most prevalent non-communicable diseases worldwide, is characterised by the progressive deterioration of the structure and mechanical properties of dental hard tissues. In human teeth, dentine is the most abundant mineralised tissue, forming the primary support material. To assess changes in the mechanical properties of dentine caused by dental caries and acid erosion, it is crucial to understand the relationship between organic and inorganic dentine components and their organisation into a 3D anisotropic structure at the nanoscale. Over the past 20 years, alterations in dentine structure caused by caries and artificial demineralisation have been reported using conventional microscopy techniques. However, due to the limited spatial resolution of these techniques, the 3D structural organisation including orientation and degree of alignment of mineralised collagen fibrils at the nanoscale, has not been fully explored. This study investigated alterations in the 3D structure of normal, carious and artificially demineralised dentine using SAXS tensor tomography (SASTT). This technique enabled the observation of differences in the local orientation of organic and inorganic components, as well as variations in local scattering intensity, resulting from natural caries and artificial demineralisation. In comparison to normal dentine, caries caused minor orientational differences of both components but had a major impact on the local X-ray scattering intensity. After artificial demineralisation of the dentine, most of the mineral was lost in the outer layers, resulting in a greater reduction in scattering intensity than that caused by caries.
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Mar 2026
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Krios I-Titan Krios I at Diamond
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Valeria
Buoli Comani
,
Omar
De Bei
,
Francesca
Pancrazi
,
Marcos
Gragera
,
Giulia
Paris
,
Marialaura
Marchetti
,
Barbara
Campanini
,
Luca
Ronda
,
Ben F.
Luisi
,
Serena
Faggiano
,
Anna Rita
Bizzarri
,
Stefano
Bettati
Diamond Proposal Number(s):
[31589]
Open Access
Abstract: To overcome iron limitation in the host, Staphylococcus aureus exploits sophisticated mechanisms to acquire this essential nutrient, particularly from hemoglobin (Hb). The bacterial hemophores IsdH and IsdB play key roles in binding Hb and extracting heme, but the structural and mechanistic differences underlying their individual contributions remain poorly defined. In this study, we dissected the molecular mechanisms by which IsdH engages Hb and mediates heme extraction, using cryo-electron microscopy, biochemical assays, and single-molecule force spectroscopy. Our structural analyses revealed pronounced conformational heterogeneity within IsdH:Hb complexes, highlighting marked flexibility in the heme-binding domain of IsdH, likely underlying its distinct functional behavior. This plasticity contrasts with the more rigid architecture of IsdB. The flexibility observed in IsdH correlates with our biochemical and biophysical findings, supporting its functional relevance. Unlike IsdB, IsdH does not display selectivity for α- or β-Hb chains and shows reduced involvement of the heme-binding domain in Hb recognition. It also follows a distinct kinetic mechanism for heme capture, which begins upon binding but proceeds more slowly than in IsdB. Finally, IsdH does not exhibit the catch bond-like behavior characteristic of IsdB, suggesting it may act in different physiological niches or conditions. Collectively, these findings highlight a distinct mode of Hb engagement by IsdH, shaped by its dynamic and flexible architecture, and provide mechanistic insight into the diversity of iron acquisition strategies employed by S. aureus.
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Dec 2025
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[23724]
Abstract: Dental caries is the most prevalent oral disease that causes structural and compositional changes of the dental hard tissues due to a chronic demineralisation (combined with possible phases of remineralisation) process. The changes can affect most important oral functions and aesthetics, as well as causing pain and discomfort. Though considerable efforts have been directed at studying natural and artificial carious lesions, most characterisations remain either constrained to 2D analyses or have been unable to achieve fine resolution in 3D due to limited field of view. To overcome this challenge, the present study combined X-ray diffraction (XRD) and scanning transmission X-ray microscopy (STXM) tomography techniques to analyse the mineral density, scattering intensity, and crystallite size in normal, carious, 30 % artificially demineralised, and 50 % artificially demineralised dentine. Combined XRD and STXM tomography was performed on the I18 beamline at Diamond Light Source, using a 15 keV monochromatic beam with 2 × 2 μm spotsize and scanning with translation steps of 2 μm, providing a reconstructed voxel size of 2 × 2 × 2 μm. Natural carious dentine showed a reduction in hydroxyapatite (HAp) crystallite size due to chronic demineralisation. This was unlike artificially demineralised dentine samples that underwent short, continuous demineralisation, which created a zone of fully demineralised dentine, near the sample surface, and a zone of partially demineralised dentine that had a reduced mineral density but an increased average crystallite size.
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May 2025
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B21-High Throughput SAXS
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Open Access
Abstract: Despite sharing ∼ 43 % sequence identity and structurally similar individual domains, botulinum neurotoxin (BoNT) serotypes A and E have differences in their properties and domain positioning. BoNT/E has a faster onset of action than BoNT/A. This difference is proposed to be due to conformational differences between BoNT/E and the other BoNT serotypes. Where most serotypes have the light chain (LC) and binding domain (BD) on opposite sides of the translocation domain (TD), BoNT/E forms a more compact shape with direct interactions between residues of the LC and BD. To elucidate the structural basis for the different properties of BoNT/A and BoNT/E, biophysical studies including molecular dynamic (MD) simulations, circular dichroism (CD) and small-angle X-ray scattering (SAXS) were applied to BoNT/A, for comparison against previous work on BoNT/E.
MD simulations at six pH values across the toxin’s activation barrier (pH ∼ 5.5), followed by one extra repeat for the pH values below 5.5, revealed a rare event at pH 5 and 5.5 where interactions between a previously identified switch region of BoNT/A and the BD were lost. This hinted at an increased freedom of movement, thus allowing the region to change from α-helical to a β-hairpin. In good agreement with previous work, CD showed a gradual and small loss of helicity as the pH decreased below pH 5.5, stabilising at pH 4.5. Combined with the relative scarcity of structural changes observed by MD in the switch region required for activity, these results may explain the slower onset of action for BoNT/A compared to BoNT/E.
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Mar 2025
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I03-Macromolecular Crystallography
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Andry Mercedes
Mavila
,
Jhon Antoni
Vargas
,
Eloy
Condori
,
Erick Giancarlo
Suclupe Farro
,
Adriano
Alves Furtado
,
Josué Manuel
López
,
Silvia Lucila
Gonzalez
,
Humberto D’muniz
Pereira
,
Jorge Luis
Marapara
,
Roger Ruiz
Paredes
,
Marianela
Cobos
,
Juan C.
Castro
,
Richard Charles
Garratt
,
Diego Antonio
Leonardo
Diamond Proposal Number(s):
[31229]
Abstract: Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg2+-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.
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Mar 2025
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Abstract: Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.0 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.
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Sep 2023
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495]
Open Access
Abstract: Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.
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Sep 2023
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I03-Macromolecular Crystallography
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Abstract: NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD± by sirtuins, PARPs, and other NAD±-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.
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Jul 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[32446]
Open Access
Abstract: Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge synthetic biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.
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May 2023
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.
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Mar 2023
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