B21-High Throughput SAXS
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Open Access
Abstract: The SINE-encoded B2 retrotransposon is an RNA Polymerase III (POL-III)-derived transcript whose expression is substantially upregulated during various cellular stress responses. Beyond retrotransposition, the B2 non-coding RNA can directly bind and repress the activity of RNA Polymerase II (POL-II), leading to a significant downregulation of transcripts during stress. Notably, our recent findings have shown that B2 is a self-cleaving ribozyme whose activity can be induced by interactions with chromatin-modifying factors through non-canonical epigenetic mechanisms that co-regulate its function across distinct chromatin-binding target loci. Here, by integrating RNA chemical probing, small-angle X-ray scattering, and 3D motif modeling, we determine structural ensemble-to-function relations for the B2 SINE ribozyme RNA. Genetic perturbations of the RNA suggest that the B2 SINE ribozyme has a well-defined secondary and dynamic tertiary structure that depends on the integrity of the critical region, which confers ribozymatic activity and repressive extent by POL-II. Using an RNA engineering approach, we examine the effects of point mutations, deletions of the main cleavage site, and deletions of the cleavage domain on the structural ensemble of the RNA. Combining this approach with in vitro and in vivo functional perturbation methods highlights the relationships between structural ensembles and various biologically relevant functional outcomes.
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Mar 2026
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Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[19435, 18258]
Open Access
Abstract: Type IV pili are long, filamentous structures that extend from bacterial cell surfaces, enabling cells to respond to changing environments and facilitating genome plasticity. Thermus thermophilus HB27 produces two different type IV pili, each exhibiting distinct structural and functional properties. Here, we combine cryo-electron tomography, mutagenesis, and AlphaFold predictions to generate hypothetical in situ models of the T. thermophilus type IV pilus assembly machinery. Using single-particle cryo-electron microscopy, we determine structures of both filament types, enabling modelling of their surface glycans. Molecular dynamics simulations further reveal the flexibility of these glycans on extrusion. Integration of the filament structures with our hypothetical model of the assembly machinery offers a framework for further dissecting T4P architecture and biogenesis.
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Mar 2026
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Justice O.
Amofa
,
Juergen
George
,
Noella A.
Okumu
,
Moses
Ohene
,
Ermias M.
Terefe
,
Stany L.
Tsomene
,
Oluwatodimu C.
Tougue
,
Ifeoluwa O.
Bejide
,
Kevin C.
Nabukeera
,
Anita Y.
Nelson
,
Carlos S. D.
Tagne
,
Patrick Y.
Osabutey
,
Aminata
Ndiaye
,
David O.
Nkwe
,
Emmanuel C.
Ohaekenyem
,
Tolulope F.
Jolaiya
,
Harrison
Banda
,
Omorede
Ikponmwosa-Eweka
,
Woutouoba
N. David
,
Blessing B.
Ekpenyong
,
Abdoulaye
Segda
,
Oudou
Diabate
,
Aliyi H.
Jarso
,
Kaddu
Arafat
,
Alyaa
Elrashedy
,
Mulatu M.
Yadeta
,
Zipporah B.
Richard
,
Isaac E.
Omara
,
Tshepang
Ndaba
,
Innocentmary I.
Ejiofor
,
Pierre F. R.
Magwell
,
Mohamed
Sedeek
,
Linda O.
Lazaro
,
Regan M.
Nyoni
,
Rossel A.
Oketch
,
Walter
Odur
,
Yaser M.
Hassan
,
Peris
Ambala
,
Courage
Chandipwisa
,
Osim P.
Bassey
,
Laurah N.
Ondari
,
Udokang G.
Jonah
,
Christelle A.
Amoussou
,
Racheal C.
Kyomukama
,
Cedric
Yamssi
,
Sunday C.
James
,
Abdoul K.
Kone
,
Oumar
Ndiaye
,
Henry
Ssenfuka
,
Agatha K.
Nyang’au
,
Yohana
Amos
,
Hakiimu
Kawalya
,
Bernard
Mware
,
Washingtone J.
Adundo
,
Vanessa B.
Ngannang-Fezeu
,
Alphonse G.
Tandja
,
Ahmed H.
Abdellatif
,
Oladokun F.
Omowumi
,
Nsubuga M.
Luutu
,
Angelo K. B.
Kouman
,
Doaa S.
Soliman
,
Nehemiah K.
Essilfie
,
James J.
Wabwile
,
Safiétou
Sankhe
,
Fatoumata G.
Fofana
,
Walid
Heiba
,
Yao
Nasser
,
Appolinaire
Djikeng
,
Eva
Akurut
,
Andrew
Walakira
,
Aurélien F. A.
Moumbock
,
Julia J.
Griese
,
Calvin
Tiengwe
,
Mama
Ndi
,
Itziar S.
Martin
,
Michel
Fodje
,
Nicolas V.
Rüffin
,
Katharina C.
Cramer
,
Jamaine
Davis
,
Emmanuel
Nji
Open Access
Abstract: Artificial intelligence is rapidly transforming structural biology and accelerating access to protein structures, yet many Africa-based scientists still lack infrastructure, training opportunities, and sustained mentorship to fully benefit. Here, we describe BioStruct-Africa’s community-driven framework integrating AlphaFold, experimental structural biology, and computational drug design to train 1000 scientists over the next decade.
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Feb 2026
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I03-Macromolecular Crystallography
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Catherine T.
Fletcher
,
Abigail A.
Mornement
,
Charlotte
Barrett
,
Peter
Canning
,
Prakash
Rucktooa
,
Sophie
Huber
,
Christopher
Cooper
,
Conor C. G.
Scully
,
Andrew S.
Dore
,
Daniel
Rohle
,
Geoffrey M. T.
Smith
,
Sarah E.
Skerratt
,
Amanda J.
Kennedy
Open Access
Abstract: Werner syndrome helicase (WRN) is a RecQ-family DNA helicase essential for genome maintenance and is a synthetic lethal target in microsatellite instability-high (MSI-H) cancers. Despite its therapeutic promise, the conformational dynamics that enable WRN to unwind DNA, and how inhibitors disrupt this activity, remains poorly understood. Here, we present crystal structures of apo WRN and WRN bound to single-stranded DNA (ssDNA), capturing key conformations in the helicase catalytic cycle. These structures reveal how WRN engages DNA through conserved polar and aromatic interactions, and how domain rearrangements, including an ordering of the aromatic-rich loop (ARL), drive directional translocation. Biochemical and biophysical data demonstrate how nucleotide and inhibitor binding remodel these conformations and suggest that known clinical inhibitors (HRO761 and VVD-133214) function by locking WRN in inactive, ‘off-DNA’ states. Resistance emerged rapidly in vitro, through acquired point mutations as well as altered WRN expression. Together, our findings provide a structural framework for the WRN structural cycle and support the development of next-generation ‘on-DNA’ inhibitors to overcome resistance.
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Jan 2026
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B21-High Throughput SAXS
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María Florencia
Pignataro
,
Natalia Brenda
Fernández
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Alba
Garay-Alvarez
,
María Florencia
Pavan
,
Rafael
Molina
,
Ines G.
Munoz
,
Julián
Grossi
,
Martín
Noguera
,
Antonella
Vila
,
Augusto E.
García
,
Hernán G.
Gentili
,
Naira Antonia
Rodríguez
,
Martín
Aran
,
Viviana
Parreño
,
Marina
Bok
,
Juan A.
Hermoso
,
Lorena Itatí
Ibañez
,
Javier
Santos
Diamond Proposal Number(s):
[35926]
Open Access
Abstract: Iron-sulfur clusters are essential cofactors for the accurate cellular function of many proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the action of the Cys desulfurase supercomplex, which is activated by the protein frataxin (FXN). The decrease of FXN expression and/or function results in Friedreich’s ataxia (FRDA).
In this work, several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity and a strong interaction with FXN. Nanobody interaction stabilized wild-type and FRDA-related FXN variants in vitro. FXN-nanobody complexes were characterized by NMR, SAXS, and X-ray crystallography. Additionally, Nanobody expression was studied in human cells. The subcellular localization, direct interaction with FXN by in situ proximity ligation assay, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. Significantly, nanobody expression did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway.
As a whole, our results suggest that nanobodies can serve as binding partners for mitochondrial FXN. However, the specific effect of the nanobodies on the conformational stability of FRDA-related FXN variants in cells should be investigated.
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Jan 2026
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I23-Long wavelength MX
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Jessica
Domenech
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Nuttawan
Pramanpol
,
Claudine
Bisson
,
Sveta E.
Sedelnikova
,
Joshua R.
Barrett
,
Abdul A. A. B.
Dakhil
,
Vitaliy
Mykhaylyk
,
Ali S.
Abdelhameed
,
Stephen E.
Harding
,
David W.
Rice
,
Patrick J.
Baker
,
Juan
Ferrer
Diamond Proposal Number(s):
[300, 1218, 24447, 31850]
Open Access
Abstract: Enzymes from salt-in halophiles are stable in conditions of low water activity with applications in chiral synthesis requiring organic solvents, yet the origins of such stability remains poorly understood. Here we describe the molecular basis of the reaction mechanism and dual NADH/NADPH-specificity of D2HDH, a 2-hydroxyacid dehydrogenase from the extreme halophile Haloferax mediterranei, an organism whose proteins have to remain active in high intracellular concentrations of KCl. Halophilic adaptations of D2HDH include the expected acidic surface and a reduction in hydrophobic surface resulting from a lower lysine content. Structure determination of crystals of D2HDH grown with KCl showed that bound K+ ions were coordinated predominantly by clusters of main chain protein carbonyl ligands, with no involvement of the numerous exposed surface carboxyls. Structural comparisons identified similar sites in other halophilic proteins suggesting that the generic use of carbonyl clusters to coordinate K+ ions may also contribute in a carboxylate-independent way to the stabilisation of the folded state of the protein in its high salt environment.
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Aug 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269, 21265]
Open Access
Abstract: Mono-ADP-ribosyl transferase (mART) proteins are secreted virulence factors produced by several human pathogens, the founding member being diphtheria toxin (DT). Pseudomonas aeruginosa can also secrete a mART toxin, known as exotoxin A (PE), but with an organization of its three functional domains (receptor, translocation, and enzymatic elements) that is opposite to DT. Two additional PE-like toxins (PLTs) have been identified from Vibrio cholerae and Aeromonas hydrophila, suggesting more PLT family members may exist. Database mining discovered six additional putative homologues, considerably extending this group of PLTs across a wide range of bacterial species. Here, we examine sequence and structural information for these new family members with respect to previously identified PLTs. The X-ray crystal structures of four new homologues show the conservation of critical features responsible for structure and function. This study shows the potential of these newly described toxins for the development of novel drug delivery platforms. Additionally, genomic analysis suggests horizontal gene transfer to account for the wide distribution of PLTs across a range of eubacteria species, highlighting the need to monitor emerging pathogens and their virulence factors.
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Mar 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[20287]
Open Access
Abstract: The Hsp70 chaperone system is capable of disassembling pathological aggregates such as amyloid fibres associated with serious degenerative diseases. Here we examine the role of the J-domain protein co-factor in amyloid disaggregation by the Hsc70 system. We used cryo-EM and tomography to compare the assemblies with wild-type DNAJB1 or inactive mutants. We show that DNAJB1 binds regularly along α-synuclein amyloid fibrils and acts in a 2-step recruitment of Hsc70, releasing DNAJB1 auto-inhibition before activating Hsc70 ATPase. The wild-type DNAJB1:Hsc70:Apg2 complex forms dense arrays of chaperones on the fibrils, with Hsc70 on the outer surface. When the auto-inhibition is removed by mutating DNAJB1 (ΔH5 DNAJB1), Hsc70 is recruited to the fibrils at a similar level, but the ΔH5 DNAJB1:Ηsc70:Apg2 complex is inactive, binds less regularly to the fibrils and lacks the ordered clusters. Therefore, we propose that 2-step activation of DNAJB1 regulates the ordered assembly of Hsc70 on the fibril. The localised, dense packing of chaperones could trigger a cascade of recruitment and activation to give coordinated, sequential binding and disaggregation from an exposed fibril end, as previously observed in AFM videos. This mechanism is likely to be important in maintaining a healthy cellular proteome into old age.
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Mar 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[29948]
Open Access
Abstract: Class I ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides under oxic conditions. The R2 subunit provides a radical required for catalysis conducted by the R1 subunit. In most R2s the radical is generated on a tyrosine via oxidation by an adjacent metal site. The discovery of a metal-free R2 defined the new RNR subclass Ie. In R2e, three of the otherwise strictly conserved metal-binding glutamates in the active site are substituted. Two variants have been found, VPK and QSK. To date, the VPK version has been the focus of biochemical characterization. Here we characterize a QSK variant of R2e. We analyse the organismal distribution of the two R2e versions and find dozens of organisms relying solely on the QSK RNR for deoxyribonucleotide production. We demonstrate that the R2eQSK of the human pathogen Gardnerella vaginalis (Bifidobacterium vaginale) modifies the active site-adjacent tyrosine to DOPA. The amount of modified protein is shown to be dependent on coexpression with the other proteins encoded in the RNR operon. The DOPA containing R2eQSK can support ribonucleotide reduction in vitro while the unmodified protein cannot. Finally, we determined the first structures of R2eQSK in the unmodified and DOPA states.
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Feb 2025
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I03-Macromolecular Crystallography
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Mara C.
Inniss
,
Sean G.
Smith
,
Dan Jun
Li
,
Benjamin
Primack
,
Dexue
Sun
,
Grace Y.
Olinger
,
Kerri-Lynn
Sheahan
,
Theresa
Ross
,
Meghan
Langley
,
Violet
Young
,
Andres
Alvarado
,
Shabnam
Davoodi
,
Jiefei
Geng
,
Michael
Schebesta
,
Michelle L.
Ols
,
Jeremy
Tchaicha
,
Jan
Ter Meulen
,
Dhruv K.
Sethi
Open Access
Abstract: Adoptive cell therapies (ACT) have shown reduced efficacy against solid tumor malignancies compared to hematologic malignancies, partly due to the immunosuppressive nature of the tumor microenvironment (TME). ACT efficacy may be enhanced with pleiotropic cytokines that remodel the TME; however, their expression needs to be tightly controlled to avoid systemic toxicities. Here we show T cells can be armored with membrane-bound cytokines with surface expression regulated using drug-responsive domains (DRDs) developed from the 260-amino acid protein human carbonic anhydrase 2 (CA2). The CA2-DRD can be stabilized in vitro and in vivo with the FDA-approved small-molecule CA2 inhibitor acetazolamide (ACZ). We develop conditional degrons using library-based screening of mutants and show characterization of one DRD using crystallography and molecular dynamics (MD) simulations. Using protein-engineering solutions to increase the valency of DRDs fused to the cargo we have developed “modulation hubs” and show tight regulation of membrane-bound cytokines IL2, IL12, IL15, IL21, IL23, and IFNα in genetically engineered T cells. Finally, CA2-DRD regulated IL12 mediates regulated efficacy in a solid tumor model. Regulation of pleotropic cytokines potentially paves the way to safely use these powerful cytokines in ACT for cancer treatment.
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Jan 2025
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