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Open Access
Abstract: Advancements in macromolecular crystallography, driven by improved sources and cryocooling techniques, have enabled the use of increasingly smaller crystals for structure determination, with microfocus beamlines now widely accessible. Initially developed for challenging samples, these techniques have culminated in advanced beamlines such as VMXm. Here, an in vacuo sample environment improves the signal-to-noise ratio in X-ray diffraction experiments, and thus enables the use of submicrometre crystals. The advancement of techniques such as microcrystal electron diffraction (MicroED) for atomic-level insights into charged states and hydrogen positions, along with room-temperature crystallography to observe physiological states via serial crystallography, has driven a resurgence in the use of microcrystals. Reproducibly preparing small crystals, especially from samples that typically yield larger crystals, requires considerable effort, as no one singular approach guarantees optimal crystals for every technique. This review discusses methods for generating such small crystals, including mechanical crushing and batch crystallization with seeding, and evaluates their compatibility with microcrystal data-collection modalities. Additionally, we examine sample-delivery methods, which are crucial for selecting appropriate crystallization strategies. Establishing reliable protocols for sample preparation and delivery opens new avenues for macromolecular crystallography, particularly in the rapidly progressing field of time-resolved crystallography.
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May 2025
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Krios V-Titan Krios V at Diamond
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Bin
Yang
,
Durga
Devalla
,
Silvia
Sonzini
,
Mikael
Boberg
,
Sashi
Gopaul
,
Monika
Sundqvist
,
Iain
Grant
,
Christopher
Jones
,
Stephanie
Brookes
,
Cindy
Weidauer
,
Eleonora
Paladino
,
Najet
Mahmoudi
,
Jason
Van Rooyen
,
Ana Gomes
Dos Santos
,
Johanna
Laru
,
Andy
Campbell
,
Lutz
Jermutus
,
Annette
Bak
Diamond Proposal Number(s):
[31098, 37220]
Abstract: Cotadutide (Cota) is a lipidated dual GLP-1 and Glucagon receptor agonist that was investigated for the treatment of various metabolic diseases, it is designed for once daily subcutaneous (SC) administration. Invasive daily injections can result in poor patient compliance with chronic disease, and here, we demonstrate an innovative strategy of encapsulating reversible cota self-assembled fibers within an in-situ forming depot of low molecular weight poly(lactic-co-glycolic) acid (LWPLGA) for sustained delivery GLP-1 and Glucagon receptor agonist with controlled burst release. This could be a suitable alternative to other sustained delivery strategies for fibrillating peptides. We investigated a range of cationic ions (Na+, Ca2+, Zn2+) and studied their influence on the secondary structure, morphology and the monomer release profile of cota fibers. Fibers forming hierarchy structures such as twisted filament and ribbons with beta sheet secondary structure resulted in better controlled burst. The subcutaneous administration of Ca2+ fiber/LWPLGA depot formulation in rats resulted in 60-fold reduction in maximum concentration (Cmax) compared with cota immediate release (IR) SC formulation and a prolonged plasma exposure over a month with plasma half-life extended from the 10 h observed with the cota daily formulation to 100 h. This extended-release formulation also maintains smaller peak and trough fluctuation within therapeutic window, and PK modelling of repeated dose indicates this formulation could enable a possible dose frequency of 14 days in rat with assumed therapeutic concentration (ratios of the maximum concentration and the trough concentration) Cmax/Ctrough window. This new long-acting injectable (LAI) method could open the door to transforming short-life peptides with sub-optimal half-life into candidates for weekly or even monthly dosing regimens, potentially leading to novel drug products with increased patient comfort.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Human diphtheria toxin-like ADP-ribosyltransferases, PARPs and tankyrases, transfer ADP-ribosyl groups to other macromolecules, thereby controlling various signaling events in cells. They are considered promising drug targets, especially in oncology, and a vast number of inhibitors have already been successfully developed. These inhibitors typically occupy the nicotinamide binding site and extend along the NAD+ binding groove of the catalytic domain. Quinazolin-4-ones have been explored as compelling scaffolds for such inhibitors and we have identified a new position within the catalytic domain that has not been extensively studied yet. In this study, we investigate larger substituents at the C-8 position and, using X-ray crystallography, we demonstrate that nitro- and diol-substituents engage in new interactions with TNKS2, improving both affinity and selectivity. Both diol- and nitro-substituents exhibit intriguing inhibition of TNKS2, with the diol-based compound EXQ-1e displaying a pIC50 of 7.19, while the nitro-based compound EXQ-2d's pIC50 value is 7.86. Both analogues impact and attenuate the tankyrase-controlled WNT/β-catenin signaling with sub-micromolar IC50. When tested against a wider panel of enzymes, the nitro-based compound EXQ-2d displayed high selectivity towards tankyrases, whereas the diol-based compound EXQ-1e also inhibited other PARPs. Compound EXQ-2d displays in vitro cell growth inhibition of the colon cancer cell line COLO 320DM, while compound EXQ-1e displays nonspecific cell toxicity. Collectively, the results offer new insights for inhibitor development targeting tankyrases and PARPs by focusing on the subsite between a mobile active site loop and the canonical nicotinamide binding site.
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Apr 2025
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B21-High Throughput SAXS
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Saishree S.
Iyer
,
Fangrui
Chen
,
Funso E.
Ogunmolu
,
Shoeib
Moradi
,
Vladimir A.
Volkov
,
Emma J.
Van Grinsven
,
Chris
Van Hoorn
,
Jingchao
Wu
,
Nemo
Andrea
,
Shasha
Hua
,
Kai
Jiang
,
Ioannis
Vakonakis
,
Mia
Potočnjak
,
Franz
Herzog
,
Benoît
Gigant
,
Nikita
Gudimchuk
,
Kelly E.
Stecker
,
Marileen
Dogterom
,
Michel O.
Steinmetz
,
Anna
Akhmanova
Diamond Proposal Number(s):
[21035]
Open Access
Abstract: Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4. We found that whereas CEP97 does not bind to microtubules directly, CP110 autonomously binds microtubule plus ends, blocks their growth, and inhibits depolymerization. Cryo-electron tomography revealed that CP110 associates with the luminal side of microtubule plus ends and suppresses protofilament flaring. CP110 directly interacts with CPAP, which acts as a microtubule polymerase that overcomes CP110-induced growth inhibition. Together, the two proteins impose extremely slow processive microtubule growth. Disruption of CP110–CPAP interaction in cells inhibits centriole elongation and increases incidence of centriole defects. Our findings reveal how two centriolar cap proteins with opposing activities regulate microtubule plus-end elongation and explain their antagonistic relationship during centriole formation.
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Mar 2025
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I03-Macromolecular Crystallography
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Open Access
Abstract: Differential scanning fluorimetry screening of the Library of Pharmacologically Active Compounds (LOPAC) identified four hits for the PRYSPRY domain of the human E3 ligase tripartite motif-containing protein 21 (TRIM21). Isothermal titration calorimetry subsequently confirmed suramin as a binder with micromolar affinity. To further investigate the binding mechanism, mouse TRIM21 was used as a structural surrogate due to its improved protein stability and high sequence similarity to the human counterpart. A crystal structure of the complex refined at 1.3 Å resolution revealed a unique binding mode, providing new avenues for targeting TRIM21 and for the development of proteolysis-targeting chimeras (PROTACs).
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Mar 2025
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Abstract: α-actinin (ACTN) is a pivotal member of the actin-binding protein family, crucial for the anchoring and organization of actin filaments within the cytoskeleton. Four isoforms of α-actinin exist: two non-muscle isoforms (ACTN1 and ACTN4) primarily associated with actin stress fibers and focal adhesions, and two muscle-specific isoforms (ACTN2 and ACTN3) localized to the Z-disk of the striated muscle. Although these isoforms share structural similarities, they exhibit distinct functional characteristics that reflect their specialized roles in various tissues. Genetic variants in α-actinin isoforms have been implicated in a range of pathologies, including cardiomyopathies, thrombocytopenia, and non-cardiovascular diseases, such as nephropathy. However, the precise impact of these genetic variants on the α-actinin structure and their contribution to disease pathogenesis remains poorly understood. This review provides a comprehensive overview of the structural and functional attributes of the four α-actinin isoforms, emphasizing their roles in actin crosslinking and sarcomere stabilization. Furthermore, we present detailed structural modeling of select ACTN1 and ACTN2 variants to elucidate mechanisms underlying disease pathogenesis, with a particular focus on macrothrombocytopenia and hypertrophic cardiomyopathy. By advancing our understanding of α-actinin’s role in both normal cellular function and disease states, this review lays the groundwork for future research and the development of targeted therapeutic interventions.
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Mar 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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I23-Long wavelength MX
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Diamond Proposal Number(s):
[32794]
Open Access
Abstract: Voltage-dependent anion channel 1 (VDAC1) is a key protein in cellular metabolism and apoptosis. Here, we present a protocol to express and purify milligram amounts of recombinant VDAC1 in Escherichia coli. We detail steps for a fluorescence polarization-based high-throughput screening assay using NADH displacement, along with procedures for thermostability, fluorescence polarization, and X-ray crystallography. In this context, we demonstrate how 2-methyl-2,4-pentanediol (MPD), a crystallization reagent, interferes with VDAC1 small-molecule binding, hindering the detection of these ligands in the crystal.
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Mar 2025
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Virginie
Will
,
Lucile
Moynie
,
Elise
Si Ahmed Charrier
,
Audrey
Le Bas
,
Lauriane
Kuhn
,
Florian
Volck
,
Johana
Chicher
,
Hava
Aksoy
,
Morgan
Madec
,
Cyril
Antheaume
,
Gaëtan L. A.
Mislin
,
Isabelle J.
Schalk
Diamond Proposal Number(s):
[33133]
Abstract: Iron is essential for bacterial growth, and Pseudomonas aeruginosa synthesizes the siderophores pyochelin (PCH) and pyoverdine to acquire it. PCH contains a thiazolidine ring that aids in iron chelation but is prone to hydrolysis, leading to the formation of 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde (IQS). Using mass spectrometry, we demonstrated that PCH undergoes hydrolysis and oxidation in solution, resulting in the formation of aeruginoic acid (AA). This study used proteomic analyses and fluorescent reporters to show that AA, dihydroaeruginoic acid (DHA), and PCH induce the expression of femA, a gene encoding the ferri-mycobactin outer membrane transporter in P. aeruginosa. Notably, the induction by AA and DHA was observed only in strains unable to produce pyoverdine, suggesting their weaker iron-chelating ability compared to that of pyoverdine. 55Fe uptake assays demonstrated that both AA-Fe and DHA-Fe complexes are transported via FemA; however, no uptake was observed for PCH-Fe through this transporter. Structural studies revealed that FemA is able to bind AA2-Fe or DHA2-Fe complexes. Key interactions are conserved between FemA and these two complexes, with specificity primarily driven by one of the two siderophore molecules. Interestingly, although no iron uptake was noted for PCH through FemA, the transporter also binds PCH-Fe in a similar manner. These findings show that under moderate iron deficiency, when only PCH is produced by P. aeruginosa, degradation products AA and DHA enhance iron uptake by inducing femA expression and facilitating iron transport through FemA. This provides new insights into the pathogen’s strategies for iron homeostasis.
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Mar 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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