Krios II-Titan Krios II at Diamond
|
Zak
Mciver
,
Didi
He
,
Jennifer
Ross
,
Marta
Cozzaglio
,
Cecilia
Piergentili
,
Aritha
Dornau
,
Natasha
Sumpner
,
Finn
Brady
,
Kathleen
Bialik
,
Thomas
Mccorvie
,
Claudia
Sissi
,
Arnaud
Basle
,
David J.
Clarke
,
Jon
Marles-Wright
Diamond Proposal Number(s):
[31827]
Open Access
Abstract: Encapsulins are self-assembling protein nanocompartments found in bacteria and archaea that encapsulate cargo enzymes to protect the cell from their toxic reaction products or intermediates. Developments in cryo-electron microscopy (cryo-EM) data processing strategies have enabled encapsulins and their cargo proteins to be investigated together in greater detail. In this study, we present the single particle cryo-EM structure of the Rhodospirillum rubrum encapsulin in both the presence and absence of its partner encapsulated ferritin (EncFtn). Single particle icosahedral reconstructions of empty and loaded encapsulins revealed a higher degree of conformational flexibility at the five-fold pore in the cargo loaded encapsulin. We applied a new non-point group averaging workflow to analyze the encapsulated ferritins within the encapsulin nanocompartment, to produce the first fully refined in situ atomic model of the EncFtn at 2.8 Å resolution. Masked 2D classification and particle subtraction demonstrate that cargo loading is heterogeneous in this recombinant complex, with the encapsulin able to house up to five of the decameric EncFtn complexes. Our data provides new insights into the dynamics and cargo arrangement in encapsulins and demonstrates an adaptable workflow for high resolution reconstruction of encapsulin cargoes.
|
Sep 2026
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[26794, 34182]
Open Access
Abstract: Obtaining reproducible and high-quality crystals is a prerequisite in X-ray crystallography to determine high resolution protein structures. Often, protein crystallization is sensitive to several factors leading to difficulties in their reproducibility and diffraction quality. A number of methods have been developed to improve crystallization. Here, we report on the strategies applied to overcome the challenges encountered during the crystallization and structure determination of a truncated soluble variant of OmamC (Rv1363c; OmamC129–261). OmamC is a homolog of mammalian cell entry (Mce) associated membrane (Mam) proteins of the lipid importing Mce complexes. OmamC is essential for the storage and utilization of lipids under starvation in mycobacteria. Heat treatment of OmamC129–261 prior to crystallization setup to remove partially unfolded protein and using the reservoir solution from the wells which successfully produced crystals as precipitant in subsequent crystallization trials (precipitant salvaging) enhanced the reproducibility and diffraction quality of OmamC129–261 crystals significantly. Heat treatment combined with precipitant salvaging is easily adaptable to any protein of interest and therefore, a valuable addition to the existing toolkit of protein crystallization improvement strategies. Furthermore, despite the AlphaFold2 predicted model providing an initial molecular replacement solution, experimental phasing was necessary highlighting its importance in determining the structure of OmamC129–261.
|
Sep 2026
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[19880]
Abstract: AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH predominantly forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AFG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function.
|
Aug 2026
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios II-Titan Krios II at Diamond
|
Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Bacteroides thetaiotaomicron (B. theta) is a model Bacteroidota of the healthy human gut microbiota and a specialist in glycan utilisation. Like other Bacteroides, B. theta has many highly regulated polysaccharide utilisation loci (PUL) that encode outer membrane (OM) TonB-dependent transporters (SusC), closely associated “lid” lipoproteins (SusD), and additional surface-exposed lipoproteins (SLPs) that bind and partially degrade specific glycans derived from host cells, diet, or other microbiota members. The canonical starch PUL products are thought to form a dynamic complex in the presence of starch. However, other PULs form stable complexes in the absence of substrate (recently named “utilisomes”), with additional surface lipoproteins tightly associated with the core SusCD complex. In this study, we characterised the B. theta dextran utilisome, with a SusCDdex core and an associated glycoside hydrolase (GHdex) and surface glycan binding protein (SBGPdex). Via X-ray crystallography we solved high-resolution structures of SBGPdex in isolation and SusDdex and GHdex bound to dextran oligosaccharides. We used isothermal titration calorimetry (ITC) to quantify ligand binding of wild type and mutant SLPs. We further used single particle cryo-EM of the catalytically inactive dextran utilisome to visualise open and closed states of the complex. Three occupied dextran binding sites were observed across SusCdex, SusDdex and GHdex, with substrate observed in both open and closed states of SusDdex. 3D variability analysis showed a minority of particles in the process of SusDdex lid closure. Together our work defines commonalities and differences across utilisomes dedicated to the import of simple glycans.
|
Jul 2026
|
|
|
|
Open Access
Abstract: Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation and the receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63 by mRNA display and characterized it biochemically and with respect to ACE2-binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 Å resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the catalytic cleft of the peptidase domain, without contacting the conserved zinc ion site. WJL-63 residues Q4, R7, R11, and R14 anchor the peptide in the binding pocket. The peptide contacts both peptidase subdomains. The upright binding mode requires an open ACE2 conformation, in contrast to small-molecule carboxypeptidase inhibitors, which typically bind to the closed conformation. One side of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells, where it exhibited binding with a KD value of 90 ± 28 nM. The ACE2 − WJL-63 complex structure provides a basis for the development of compounds that modulate ACE2 conformation and for the development of imaging agents for visualization of ACE2, including fluorescence or electron microscopy and positron emission tomography (PET).
|
Apr 2026
|
|
I22-Small angle scattering & Diffraction
|
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.
|
Mar 2026
|
|
Krios I-Titan Krios I at Diamond
|
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.
|
Dec 2025
|
|
I18-Microfocus Spectroscopy
|
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.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
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.
|
Mar 2025
|
|
B21-High Throughput SAXS
|
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.
|
Mar 2025
|
|
