B21-High Throughput SAXS
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Diamond Proposal Number(s):
[34342, 35585]
Open Access
Abstract: The interaction of the surfactant-like peptide (SLP) R3L12 bearing three cationic arginine residues with model liposomes is investigated in aqueous solution at various pH values, under conditions for which the SLP self-assembles into nanotubes. The structure of liposomes of model anionic lipid DPPG [1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)], or zwitterionic lipid DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine] is probed using small-angle X-ray scattering and cryogenic-transmission electron microscopy. The unilamellar vesicles of DPPG are significantly restructured in the presence of R3L12, especially at low pH, and multilamellar vesicles of DPPE are also restructured under these conditions. The SLP promotes the release of cargo encapsulated in the vesicles as probed by calcein fluorescence, with notably higher release for anionic DPPG vesicles. Laurdan fluorescence experiments to probe membrane fluidity (lipid chain ordering) show that R3L12 destabilizes the lipid gel phase, especially for anionic DPPG. This model nanotube-forming SLP has promise as a pH-sensitive release system for vesicle-encapsulated cargo.
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Oct 2024
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B24-Cryo Soft X-ray Tomography
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Diamond Proposal Number(s):
[30471, 33090]
Open Access
Abstract: Iron is a crucial element integral to various fundamental biological molecular mechanisms, including magnetosome biogenesis in magnetotactic bacteria (MTB). Magnetosomes are formed through the internalization and biomineralization of iron into magnetite crystals. However, the interconnected mechanisms by which MTB uptake and regulate intracellular iron for magnetosome biomineralization remain poorly understood, particularly at the single-cell level. To gain insights we employed a holistic multiscale approach, i.e., from elemental iron species to bacterial populations, to elucidate the interplay between iron uptake dynamics and magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 under near-native conditions. We combined a correlative microscopy approach integrating light and X-ray tomography with analytical techniques, such as flow cytometry and inductively coupled plasma spectroscopy, to evaluate the effects of iron and oxygen availability on cellular growth, magnetosome biogenesis, and intracellular iron pool in MSR-1. Our results revealed that increased iron availability under microaerobic conditions significantly promoted the formation of longer magnetosome chains and increased intracellular iron uptake, with a saturation point at 300 μM iron citrate. Beyond this threshold, additional iron did not further extend the magnetosome chain length or increase total intracellular iron levels. Moreover, our work reveals (i) a direct correlation between the labile Fe2+ pool size and magnetosome content, with higher intracellular iron concentrations correlating with increased magnetosome production, and (ii) the existence of an intracellular iron pool, distinct from magnetite, persisting during all stages of biomineralization. This study offers insights into iron dynamics in magnetosome biomineralization at a single-cell level, potentially enhancing the industrial biomanufacturing of magnetosomes.
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Oct 2024
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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[27906, 37100]
Abstract: The storage and distribution of vital protein therapeutics presents several complex challenges. Many medicines and vaccines need stable, temperature-controlled environments and chemical additives (excipients) such as preservatives to keep them effective and safe for use.
This requires cold storage infrastructure and reliable energy sources which not only puts the responsibility on the user but causes accessibility and affordability challenges, especially in developing countries where resources are limited.
Fig. 1
Fig. 1
Now researchers from the UK Universities of Manchester, Glasgow and Warwick have designed the world’s first hydrogel technology for the storage and distribution of crucial medicines and other biopharmaceuticals without the need for refrigeration or chemical additives. The aim is to provide more robust and equitable storage and delivery systems, benefitting everyone worldwide.
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Oct 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[31827]
Open Access
Abstract: Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the “ubiquitination zone.” Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.
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Oct 2024
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Yufei
Xiang
,
Jialu
Xu
,
Briana L.
Mcgovern
,
Anna
Ranzenigo
,
Wei
Huang
,
Zhe
Sang
,
Juan
Shen
,
Randy
Diaz-Tapia
,
Ngoc Dung
Pham
,
Abraham J. P.
Teunissen
,
M. Luis
Rodriguez
,
Jared
Benjamin
,
Derek J.
Taylor
,
Mandy M.t.
Van Leent
,
Kris M.
White
,
Adolfo
García-Sastre
,
Peijun
Zhang
,
Yi
Shi
Abstract: Pathogens constantly evolve and can develop mutations that evade host immunity and treatment. Addressing these escape mechanisms requires targeting evolutionarily conserved vulnerabilities, as mutations in these regions often impose fitness costs. We introduce adaptive multi-epitope targeting with enhanced avidity (AMETA), a modular and multivalent nanobody platform that conjugates potent bispecific nanobodies to a human immunoglobulin M (IgM) scaffold. AMETA can display 20+ nanobodies, enabling superior avidity binding to multiple conserved and neutralizing epitopes. By leveraging multi-epitope SARS-CoV-2 nanobodies and structure-guided design, AMETA constructs exponentially enhance antiviral potency, surpassing monomeric nanobodies by over a million-fold. These constructs demonstrate ultrapotent, broad, and durable efficacy against pathogenic sarbecoviruses, including Omicron sublineages, with robust preclinical results. Structural analysis through cryoelectron microscopy and modeling has uncovered multiple antiviral mechanisms within a single construct. At picomolar to nanomolar concentrations, AMETA efficiently induces inter-spike and inter-virus cross-linking, promoting spike post-fusion and striking viral disarmament. AMETA’s modularity enables rapid, cost-effective production and adaptation to evolving pathogens.
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Oct 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25413]
Open Access
Abstract: Serine β-lactamases inactivate β-lactam antibiotics in a two-step mechanism comprising acylation and deacylation. For the deacylation step, a water molecule is activated by a conserved glutamate residue to release the adduct from the enzyme. The third-generation cephalosporin ceftazidime is a poor substrate for the class A β-lactamase BlaC from Mycobacterium tuberculosis but it can be hydrolyzed faster when the active site pocket is enlarged, as was reported for mutant BlaC P167S. The conformational change in the Ω-loop of the P167S mutant displaces the conserved glutamate (Glu166), suggesting it is not required for deacylation of the ceftazidime adduct. Here, we report the characterization of wild type BlaC and BlaC E166A at various pH values. The presence of Glu166 strongly enhances activity against nitrocefin but not ceftazidime, indicating it is indeed not required for deacylation of the adduct of the latter substrate. At high pH wild type BlaC was found to exist in two states, one of which converts ceftazidime much faster, resembling the open state previously reported for the BlaC mutant P167S. The pH-dependent switch between the closed and open states is caused by the loss at high pH of a low-barrier hydrogen bond, a proton shared between Asp172 and Asp179. These results illustrate how readily shifts in substrate specificity can occur as a consequence of subtle changes in protein structure.
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Oct 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20303]
Open Access
Abstract: Ene reductases (EREDs) catalyze asymmetric reduction with exquisite chemo-, stereo-, and regioselectivity. Recent discoveries led to unlocking other types of reactivities toward oxime reduction and reductive C–C bond formation. Exploring nontypical reactions can further expand the biocatalytic knowledgebase, and evidence alludes to yet another variant reaction where flavin mononucleotide (FMN)-bound ERs from the old yellow enzyme family (OYE) have unconventional activity with α,β-dicarbonyl substrates. In this study, we demonstrate the nonconventional stereoselective monoreduction of α,β-dicarbonyl to the corresponding chiral hydroxycarbonyl, which are valuable building blocks for asymmetric synthesis. We explored ten α,β-dicarbonyl aliphatic, cyclic, or aromatic compounds and tested their reduction with five OYEs and one nonflavin-dependent double bond reductase (DBR). Only GluER reduced aliphatic α,β-dicarbonyls, with up to 19% conversion of 2,3-hexanedione to 2-hydroxyhexan-3-one with an R-selectivity of 83% ee. The best substrate was the aromatic α,β-dicarbonyl 1-phenyl-1,2-propanedione, with 91% conversion to phenylacetylcarbinol using OYE3 with R-selectivity >99.9% ee. Michaelis–Menten kinetics for 1-phenyl-1,2-propanedione with OYE3 gave a turnover kcat of 0.71 ± 0.03 s–1 and a Km of 2.46 ± 0.25 mM. Twenty-four EREDs from multiple classes of OYEs and DBRs were further screened on 1-phenyl-1,2-propanedione, showing that class II OYEs (OYE3-like) have the best overall selectivity and conversion. EPR studies detected no radical signal, whereas NMR studies with deuterium labeling indicate proton incorporation at the benzylic carbonyl carbon from the solvent and not the FMN hydride. A crystal structure of OYE2 with 1.5 Å resolution was obtained, and docking studies showed a productive pose with the substrate.
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Oct 2024
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Alena
Kroupova
,
Valentina A.
Spiteri
,
Zoe J.
Rutter
,
Hirotake
Furihata
,
Darren
Darren
,
Sarath
Ramachandran
,
Sohini
Chakraborti
,
Kevin
Haubrich
,
Julie
Pethe
,
Denzel
Gonzales
,
Andre J.
Wijaya
,
Maria
Rodriguez-Rios
,
Manon
Sturbaut
,
Dylan M.
Lynch
,
William
Farnaby
,
Mark A.
Nakasone
,
David
Zollman
,
Alessio
Ciulli
Diamond Proposal Number(s):
[26793, 35324, 33832, 38813]
Open Access
Abstract: The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.
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Oct 2024
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Krios III-Titan Krios III at Diamond
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Ana
Crespillo-Casado
,
Prathyush
Pothukuchi
,
Katerina
Naydenova
,
Matthew C. J.
Yip
,
Janet M.
Young
,
Jerome
Boulanger
,
Vimisha
Dharamdasani
,
Ceara
Harper
,
Pierre-Mehdi
Hammoudi
,
Elsje G.
Otten
,
Keith
Boyle
,
Mayuri
Gogoi
,
Harmit S.
Malik
,
Felix
Randow
Diamond Proposal Number(s):
[31336]
Open Access
Abstract: Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213’s dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.
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Oct 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Attila
Vasas
,
Lisa
Ivanschitz
,
Balázs
Molnár
,
Árpád
Kiss
,
Lisa
Baker
,
Andrea
Fiumana
,
Alba
Macias
,
James B.
Murray
,
Emma
Sanders
,
Neil
Whitehead
,
Roderick E.
Hubbard
,
Carine
Saunier
,
Elodie
Monceau
,
Anne Marie
Girard
,
Marion
Rousseau
,
Maia
Chanrion
,
Didier
Demarles
,
Olivier
Geneste
,
Csaba
Weber
,
Elodie
Lewkowicz
,
Andras
Kotschy
Diamond Proposal Number(s):
[5791, 6001, 5067]
Abstract: Inhibition of ubiquitin-specific protease 7, USP7, has been proposed as a mechanism to affect many disease processes, primarily those implicated in oncology. The bound crystal structure of a published high-throughput screening hit with low-micromolar affinity for USP7 identified three regions of the compound for structure-guided optimization. Replacing one side of the compound with different aromatic moieties gave little improvement in affinity, and the central piperidine could not be improved. However, the binding site for the other side of the compound was poorly defined in the crystal structure, which suggested a wide variety of synthetically accessible options for optimization. These were assessed by screening reaction mixtures that introduced different substituents to this other side. Subsequent optimization led to a compound with low-nanomolar affinity for USP7, which showed target engagement in tumors, was tolerated in mice, and showed efficacy in xenograft models.
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Oct 2024
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