I24-Microfocus Macromolecular Crystallography
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Marina
Lucic
,
Johan
Glerup
,
Pierre
Aller
,
Danny
Axford
,
Nicholas
Devenish
,
Jaehyun
Park
,
Anastasiia
Shilova
,
Arturo
Landeros De La Isla
,
Richard W.
Strange
,
Tiankun
Zhou
,
Robin L.
Owen
,
Jonathan A. R.
Worrall
,
Michael A.
Hough
Diamond Proposal Number(s):
[19458, 28583]
Open Access
Abstract: Metalloenzymes containing a heme cofactor catalyse a wide range of oxidative reactions critical to life. Understanding the structure and electronic states of the heme across the catalytic cycle is essential in understanding the oxidative chemistry performed on the substrate. This work demonstrates in crystallo manipulation of the heme-iron oxidation state in a B-type dye-decolourizing peroxidase from Streptomyces lividans (DtpB) using multiple, complementary, serial crystallography approaches. Fixed-target drop-on-chip serial femtosecond crystallography (SFX) together with dose-resolved serial synchrotron crystallography (SSX) allowed DtpB to be driven between multiple iron oxidation states. Drop-on-chip addition of hydrogen peroxide with fixed-target SFX is used to generate a ferryl [Fe(IV)=O] species, while the X-ray-driven approach modulates the iron oxidation state, with an apparent two-electron reduction leading to a return to a ferric state. The formation and dose response of the Fe(IV)—O state is highly variable between the chemically identical heme groups of the DtpB hexamer, highlighting the importance of understanding the effect of the crystalline lattice on observed changes in time- and dose-resolved crystallography.
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May 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31800]
Open Access
Abstract: With the meteoric rise in interest in GLP-1 and GIP analogue peptides in recent years, there is a drive for the use of alternative purification techniques to alleviate processing bottlenecks and reduce the cost of peptide manufacturing. However, a lack of reported crystal structures for this class of peptides has hindered molecular-scale understanding of GLP-1/GIP analogue peptide crystallization, particularly related to acylated peptides. This paper therefore reports what is believed to be the first crystal structure of a GLP-1 and GIP analogue lipopeptide. Crystals obtained using a microseed matrix-screening protocol diffracted to ≤1.6 Å resolution in space group P43, with unit-cell parameters a = b = 64.66, c = 11.42 Å. Model building and the resultant structural analysis reveals that the predominantly helical peptide forms a uniquely porous spiral crystal structure composed of clockwise-ascending monomers in a square pattern, with aromatic C⋯H—π interactions around Phe22 forming the primary crystal contact between neighbouring square motifs.
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Apr 2026
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[32728]
Open Access
Abstract: Ferritins play a key role in iron management in organisms from all kingdoms of life. Excess iron is sequestered in mineral form within the hollow protein shell and can be liberated when supply becomes restricted. The protein consists of 24 isostructural monomeric units that pack with 4-, 3-, and 2-fold symmetry. Channels through the protein coat at the 3-fold axes of ferritins localised in the cytosol of animal cells contain a strictly conserved LCDFXEX ‘twin carboxylate’ motif, and have been shown to be the major iron entry route to animal ferritins, facilitating access to the H-chain intra-subunit catalytic ferroxidase centre. In the ferritin localised to the mitochondria of animals, there is natural variation within the residues lining this channel, such that the Asp residue of the twin carboxylate motif (Asp131) is not strictly conserved. Here we report X-ray crystallographic and solution kinetic studies of the properties of D131N variants of H-chain and mitochondrial ferritins. X-ray structures revealed significant perturbation of metal binding at the three-fold channels and ferroxidase centres of H-chain ferritin, but a relatively minor effect on mitochondrial ferritin. Likewise, kinetic data showed that rapid Fe2+ uptake was abolished in the D131N variant of H-chain ferritin, but less severely impacted in the equivalent variant of mitochondrial ferritin. Differences were also observed in rates of mineralisation and extent of iron release in the D131N variants of the two ferritins. The implications for the physiological role of mitochondrial versus cytosolic ferritin are discussed.
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Apr 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The malarial parasite, Plasmodium falciparum (Pf), utilizes aminopeptidases in the breakdown of hemoglobin-derived oligopeptides to release amino acids for protein synthesis during growth and asexual reproduction of erythrocytic stages of the parasite. However, a N-terminal peptide bond that involves proline is difficult to hydrolyze. Aminopeptidase P (APP) is capable of cleaving peptide bonds with proline in the second position. Inhibition of PfAPP is therefore an attractive strategy for developing therapeutics for the treatment of malaria by limiting the supply of amino acids at the erythrocytic stage. We employed the structure-activity relationship of an existing APP inhibitor, apstatin, to design a more potent PfAPP inhibitor by introducing a hydroxamic acid metal-binding group in place of the amino-alcohol of apstatin, and an aromatic P4′ moiety. A hydroxamic tetrapeptide with phenylalanine at P4′ (6d) greatly increased the inhibitory potency (apstatin Ki, 16 μM; 6d, Ki 685 nM). Replacing the P3′ proline of 6d with a 2-substituted piperidine (6e) further improved the potency (Ki, 24 nM). Crystal structure analysis of PfAPP in complex with 6d and 6e showed binding at the active site with coordination of the hydroxamic acid metal binding group to the di-metal center, and several protein-inhibitor interactions involving domains II and III. A comparison of PfAPP-6e with human APP1 indicated that the P4′ phenylalanine drives inhibitor potency and selectivity towards PfAPP, by forming an interaction with Tyr617 of the adjacent monomer within the dimer. The details presented here should be useful for the future design of potent and selective PfAPP inhibitors.
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Mar 2026
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Serial synchrotron crystallography (SSX) enables structure determination from microcrystals under near-physiological, room-temperature conditions but is limited in part due to the inevitable onset of radiation damage. The ability to reduce the absorbed dose while retaining, or even improving, data quality is an attractive means of mitigating this limitation. Advances in detector technology have made the use of high-energy X-rays a routine approach in MX, improving diffraction efficiency and enhancing overall data quality. Here, we systematically evaluate low-dose SSX data collected at five different X-ray energies from 12.4 to 25 keV using a CdTe Eiger2 detector while maintaining a constant dose. Higher photon energies increased the mean diffracted intensity and signal-to-noise ratio per unit dose, and facilitated higher-resolution structure determination, even with limited crystal numbers. These findings highlight the advantages of high-energy X-rays and provide practical guidance for optimizing SSX experiments in probing protein dynamics.
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Mar 2026
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I24-Microfocus Macromolecular Crystallography
Detectors
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John
Matheson
,
Danny
Axford
,
Anna
Bergamaschi
,
Maria
Carulla
,
Nicholas
Devenish
,
Noemi
Frisina
,
Viktoria
Hinger
,
Vadym
Kedych
,
Christopher
Lane
,
Aldo
Mozzanica
,
Eva
Gimenez-Navarro
,
James
O'Hea
,
Dominic
Oram
,
Robin L.
Owen
,
David
Perl
,
Adam
Prescott
,
Bernd
Schmitt
,
Shane
Scully
,
Adam
Taylor
,
Gary
Yendell
,
Graeme
Winter
Open Access
Abstract: A Jungfrau-1M detector has undergone testing at Diamond Light Source. The Jungfrau series of detectors from PSI use integration and adaptive gain, to offer very high frame rate and dynamic range, suitable for high-flux and time-resolved measurements. They are becoming more widely used, to take advantage of increasing light source brightness. We report on our experiences in testing the performance of a Jungfrau-1M without illumination, with a laboratory X-ray tube and on a microfocus beamline. The Jungfrau-1M was found to be able to resolve single photons in the laboratory and on the beamline. It was confirmed that range switching from high to intermediate gain is associated with a discontinuity in the detector response. Two methods of dark frame subtraction were compared for their effect on minimizing this discontinuity. The Jungfrau-1M was found to be very effective for recording macromolecular crystallography diffraction patterns, with no apparent detriment from the discontinuity. The Diamond machine will be upgraded in 2028–9 and will operate at significantly higher flux than at present, necessitating increased use of integrating detectors, such as Jungfrau, in the future.
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Mar 2026
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[38316]
Open Access
Abstract: The DUF4465 family (DUF, domain of unknown function) contains more than 1000 members distributed across eight bacterial clades with species from diverse microenvironments including various gut microbiomes, hydrothermal vents, and soil. In the gut commensal Bacteroides thetaiotaomicron (B. theta), DUF4465 containing proteins act as high-affinity B12–binding proteins that scavenge this cofactor to ensure bacterial survival. Such B12 capture is essential for bacteria that have lost the ability to synthesize B12 de novo. This raises the question of whether B12-binding is ubiquitous across this family of proteins. Here, we show that B12-binding is a recurrent function of eight distantly related members of the DUF4465 family. It is reasonable to conclude that B12-binding is a common function of most DUF4465 proteins. These results establish DUF4465 as a structurally conserved family of augmented β-jellyroll B12-binding proteins with widespread roles in microbial competition for this essential cofactor.
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Mar 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948, 32736]
Open Access
Abstract: The acquisition of vitamin B12 and related cobamides is a key determinant for the fitness of Bacteroidota in the gut. Depending on the species, this uptake process relies on one to four transport systems centered on conserved core outer membrane (OM) complexes composed of the TonB-dependent transporter BtuB and the surface-exposed lipoprotein BtuG. Additionally, the surface-exposed lipoprotein BtuH, although not tightly associated with the BtuBG complex, contributes to cobamide uptake and provides a fitness advantage. Here, we report the functional and structural characterization of BtuJ1 from Bacteroides thetaiotaomicron (B. theta), an additional surface-exposed lipoprotein in B12 uptake loci. BtuJ1 binds vitamin B12 and cobinamide (an intermediate in B12 biosynthesis) with low nM affinity, conferring a fitness advantage in B12-limited environments. Regardless of B12 availability, BtuJ1 is the most abundant of the B12-transport components encoded by B. theta. Under B12-replete conditions, BtuJ1 binds the vitamin, generating a readily available pool for transfer to the core BtuBG transport systems during periods of B12 depletion as demonstrated by in vitro and in vivo B12 transfer experiments. Together, these findings expand the known functionalities of the diverse accessory OM proteins employed by Bacteroidota and underscore the sophisticated strategies these human gut commensals use to secure vitamin B12 in the competitive environment of the human gut.
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Mar 2026
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I24-Microfocus Macromolecular Crystallography
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James B.
Shaum
,
Miquel
Muñoz I Ordoño
,
Erica A.
Steen
,
Daniela V.
Wenge
,
Hakyung
Cheong
,
Jordan
Janowski
,
Moritz
Hunkeler
,
Eric M.
Bilotta
,
Zoe J.
Rutter
,
Paige A.
Barta
,
Abby M.
Thornhill
,
Natalia
Milosevich
,
Lauren M.
Hargis
,
Timothy R.
Bishop
,
Trever R.
Carter
,
Bryce
Da Camara
,
Matthias
Hinterndorfer
,
Lucas
Dada
,
Wen-Ji
He
,
Fabian
Offensperger
,
Hirotake
Furihata
,
Sydney R.
Schweber
,
Charlie
Hatton
,
Yanhe
Wen
,
Benjamin F.
Cravatt
,
Keary M.
Engle
,
Katherine A.
Donovan
,
Bruno
Melillo
,
Seiya
Kitamura
,
Alessio
Ciulli
,
Scott A.
Armstrong
,
Eric S.
Fischer
,
Georg E.
Winter
,
Michael A.
Erb
Diamond Proposal Number(s):
[35324]
Open Access
Abstract: Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While the discovery of heterobifunctional CIPs is expedited by rational design strategies, molecular glues have relied predominantly on serendipity. We hypothesized that preexisting ligands could be systematically decorated with chemical modifications to discover compounds that recruit proteins to a composite protein–ligand interface. Using sulfur(VI) fluoride exchange-based high-throughput chemistry (HTC) to install 3,163 structurally diverse building blocks onto ENL (eleven-nineteen leukemia) and BRD4 (bromodomain-containing protein 4) ligands, we screened each analog for degrader activity. This revealed dHTC1, an ENL degrader that recruits CRL4CRBN complex through an extended interface of protein–protein contacts and only engages CRBN after pre-forming the ENL:dHTC1 complex. We also identified dHTC3, a molecular glue that selectively dimerizes BRD4 bromodomain 1 to SCFFBXO3, an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and new effectors for proximity pharmacology.
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Feb 2026
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Mariska
De Munnik
,
Amelia
Brasnett
,
Tiankun
Zhou
,
William
Myers
,
Yicheng
Wang
,
Kuntal
Chatterjee
,
Anthony
Tumber
,
Stephen A.
Marshall
,
Philipp S.
Simon
,
Pierre
Aller
,
Anastasiia
Shilova
,
Danny
Axford
,
Hiroki
Makita
,
Daniel W.
Paley
,
Vandana
Tiwari
,
Alexander T.
Stead
,
Sebastian
Dehe
,
Humberto
Sanchez
,
Daniel J.
Rosenberg
,
Roberto
Alonso-Mori
,
Asmit
Bhowmick
,
Junko
Yano
,
Vittal K.
Yachandra
,
Jaehyun
Park
,
Sehan
Park
,
Allen M.
Orville
,
Lennart
Brewitz
,
Jan F.
Kern
,
Christopher J.
Schofield
,
Patrick
Rabe
Diamond Proposal Number(s):
[32727, 31353]
Open Access
Abstract: Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O2 surrogate nitric oxide imply O2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O2 is the sole oxygen source in alcohol products, as shown by 18O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.
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Feb 2026
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