Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[22238]
Open Access
Abstract: Respiratory complex I is a multi-subunit energy-transducing membrane enzyme essential for mitochondrial and cellular energy metabolism. It couples NADH oxidation and ubiquinone-10 (Q10) reduction to the concomitant pumping of four protons to generate the proton-motive force that powers oxidative phosphorylation. Despite recent advances in structural knowledge of complex I, many mechanistic aspects including the reactive binding poses of Q10, how Q10 reduction initiates the proton transfer cascade, and how protons move through the membrane domain, remain unclear. Here, we use electron cryomicroscopy to determine structures of mammalian complex I, reconstituted into phospholipid nanodiscs containing exogenous Q10 and reduced by NADH, to global resolutions of 2.0 to 2.6 Å. Two conformations of a reduced Q10H2 molecule are observed, fully inserted into the Q-binding channel in the turnover-relevant closed state. By comparing the quinone species bound in oxidised and reduced complex I structures, paired with molecular dynamics simulations to investigate the charge states of key surrounding residues, we propose a series of substrate binding poses that Q10 transits through for reduction. Our highly hydrated structures exhibit near-continuous proton-transfer connections along the length of the membrane domain, enabling comparisons between them to assist in identifying the proton-transfer control points that are essential to catalysis.
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Mar 2026
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Donghwi
Ko
,
Raili
Ruonala
,
Alexandre
Faille
,
Eva
Hellmann
,
Hanna
Help
,
Huili
Liu
,
Ronni
Nielsen
,
Anders
Haakonsson
,
Nuria
De Diego
,
Anja
Paatero
,
Mariia V.
Shcherbii
,
Karolina
Stefanowicz
,
Sanja
Ćavar Zeljković
,
Tine
Drud Lundager Rasmussen
,
Ondrej
Novak
,
Zsuzsanna
Bodi
,
Gugan
Eswaran
,
Brecht
Wybouw
,
Matthieu
Bourdon
,
Cristina
Urbez
,
Xiaonan
Liu
,
Kari
Salokas
,
Tiina
Öhman
,
Tanya
Waldie
,
Petri
Törönen
,
Sedeer
El-Showk
,
Martin
Balcerowicz
,
Fabrice
Besnard
,
Xiaomin
Liu
,
Patrick
Perkins
,
Serina
Mazzoni-Putman
,
Julia P.
Vainonen
,
Maija
Sierla
,
Mikko J.
Frilander
,
Susanne
Mandrup
,
Teva
Vernoux
,
Karin
Ljung
,
Alejandro
Ferrando
,
Miguel A.
Blazquez
,
Liisa
Holm
,
Rupert
Fray
,
Markku
Varjosalo
,
Ottoline
Leyser
,
Ville O.
Paavilainen
,
Ari Pekka
Mähönen
,
Anna
Stepanova
,
Jose
Alonso
,
Steffen
Heber
,
Robert
Malinowski
,
Finn
Kirpekar
,
Alan J.
Warren
,
Ykä
Helariutta
Diamond Proposal Number(s):
[37678]
Abstract: Polyamines are often associated with ribosomes and are thought to stabilize their integrity. In Arabidopsis, the polyamine thermospermine (tSpm) affects xylem cell fate. tSpm induces translation of SUPPRESSOR-OF-ACAULIS51 (SAC51) and SAC51-LIKEs (SACLs), which inhibit heterodimerization of the xylem development proteins LONESOME-HIGHWAY (LHW) and TARGET-OF-MONOPTEROS5. Here, we report a methyltransferase, OVERACHIEVER, that methylates the peptidyl transferase center of the 25S ribosomal RNA (rRNA). Residue m3U2952 promotes functional tSpm binding to a specific site connecting the P-site transfer RNA (tRNA) with rRNA residues in the peptidyl transferase center. This interaction enhances the translation of SACLs but inhibits that of LHW. Our study uncovers the dependency between a conserved rRNA base methylation and a polyamine in orchestrating cell fate decisions, highlighting a role for the ribosome chemical landscape in translational regulation.
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Feb 2026
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B21-High Throughput SAXS
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[24557, 39224]
Open Access
Abstract: Histone variants define distinct chromatin states by modulating the biophysical properties of nucleosomes. Variants play a particularly important role in the parasitic protist Trypanosoma brucei, which has unusual chromatin and lacks a canonical repressive heterochromatin system. Instead, T. brucei utilizes specialized divergent histone variants H3.V and H4.V. However, the biochemical basis of their repressive functions is unknown. Here, we determined the structure of the H3.V-H4.V nucleosome core particle and biochemically characterized variant-containing nucleosomes and nucleosome arrays, probing their unique properties. We discovered that surprisingly for repressive-state nucleosomes, H3.V promotes pronounced DNA splaying, largely via its N-terminal tail region, while retaining overall stability that is comparable to canonical nucleosomes. In contrast, H4.V exhibits near-identical binding to DNA but mediates a slight increase in histone octamer stability. The surface of the H3.V-H4.V nucleosome is altered and provides a differential platform for chromatin-binding proteins, linking the variants to parasite pathogenicity.
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Feb 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[29493]
Open Access
Abstract: During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg does not prevent cyclisation or pilus formation, which remains structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlates with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolish conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membrane’s inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation is rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.
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Feb 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[32359]
Open Access
Abstract: Atkinsonella hypoxylon virus (AhV) is a fungi-infecting betapartitivirus and the typical member of the Partitiviridae, a family of persistent viruses that infect a broad range of organisms. Partitiviruses have been largely overlooked following their designation as cryptic viruses. However, evidence is accumulating that they play an important role in the ecology of their hosts. Since the capsid proteins of partitiviruses have been implicated in virus–host interactions, exploring their structural biology may give clues into the evolution, horizontal transmission and host adaptation of partitiviruses. The capsid of AhV shares the same organization of other partitiviruses with 60 dimeric capsid protein protomers arranged with T=1 icosahedral symmetry. The structure, determined by cryo-electron microscopy to 2.4 Å, shows that AhV has a unique iteration on the protrusion domain with an extensive network of hydrophobic interactions among equivalent interdigitating loops at the dimerization interface. AhV also shares a conserved helical core in the shell domain, which we extend to all genera of the recognized partitiviruses using protein structure prediction. The helical core appears to be a conserved element of the picobirnavirus lineage of capsid protein folds and provides a template onto which various elaborations of the protrusion domain have evolved. The involvement of the protrusion in virus–host interactions has previously been proposed, and our findings provide evidence of a structural device enabling capsid protein diversification during the evolution of the Partitiviridae.
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Jan 2026
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Double-stranded (ds)RNA viruses replicate and transcribe their genome within a proteinaceous viral capsid to evade host cell defenses. While Reovirales members use conservative transcription, most dsRNA viruses, including cystoviruses, utilize semi-conservative transcription, in which a newly synthesized positive strand replaces the parental positive strand, which is released as mRNA. Here, we visualize semi-conservative transcription activation in cystovirus ɸ6 double-layered particles using cryogenic electron microscopy. We observe nucleotide-triggered disassembly of the domain-swapped outer capsid layer, subsequent expansion of the inner capsid layer, and stepwise assembly of transcription complexes at the opposing poles of the spooled dsRNA genome. These complexes consist of the viral polymerases embedded into a triskelion formed by the minor protein P7, which we show as essential for continuous transcription. The packaging hexamers proximal to the transcription sites channel the viral mRNA exit. Our results define the complex molecular pathway from the quiescent state to activated semi-conservative transcription.
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Jan 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[38262]
Open Access
Abstract: Pseudomonas putida is a plant-beneficial rhizobacterium that encodes multiple type-VI secretion systems (T6SS) to outcompete phytopathogens in the rhizosphere. Among its antibacterial effectors, Tke5 (a member of the BTH_I2691 protein family) is a potent pore-forming toxin that disrupts ion homeostasis without causing considerable membrane damage. Tke5 harbours an N-terminal MIX domain, which is required for T6SS-dependent secretion in other systems. Many MIX domain-containing effectors require T6SS adaptor proteins (Tap) for secretion, but their molecular mechanisms of adaptor-effector binding remain elusive. Here, we report the 2.8 Å cryo-EM structure of the Tap3-Tke5 complex of P. putida strain KT2440, providing structural and functional insights into how effector Tke5 is recruited by its cognate adaptor protein Tap3. Functional dissection shows that the α-helical region of Tke5 is sufficient to kill intoxicated bacteria, while its β-rich region likely contributes to target membrane specificity. These findings delineate a mechanism of BTH_I2691 proteins for Tap recruitment and toxin activity, contributing to our understanding of a widespread yet understudied toxin family.
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Jan 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[37115]
Open Access
Abstract: α7 nicotinic receptors are neurotransmitter-gated ion channels involved in neurological and inflammatory diseases. Ligands acting on its neurotransmitter binding site and on the channel domain of α7 have been extensively developed, yielding a wide range of orthosteric effectors and allosteric positive modulators. Here, we present the functional and structural characterization of two camelid antibody fragments, or nanobodies, F1 and E6, that inhibit α7 activity by acting as negative allosteric modulators, an underrepresented class of ligands. Cryo-EM structures of the nanobodies in complex with α7 show that both nanobodies form a pentameric bundle at the apex of the receptor, each nanobody interacting through a conserved set of residues at α7 subunit interfaces. Electrophysiological experiments suggest that E6 inhibits the activity of α7 by stabilizing its resting conformation, and that internanobodies interactions are key to its activity. Those two nanobodies expand the toolbox for human α7 modulation, opening new possibilities for its pharmacological control with far reaching potentialities in clinics.
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Jan 2026
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Caliciviruses are important human and animal pathogens that cause varying clinical signs including gastroenteritis, respiratory illness, and hepatitis. Despite the availability of numerous calicivirus structures, relatively little is known about the mechanisms of capsid assembly and stability, or about genome packaging. Here we present the atomic structure of the RHDV virion and several related non-infectious virus-like particles, determined using cryo-EM at 2.5-3.3 Å resolution. The inherent molecular switch, responsible for the conformational flexibility of the capsid protein VP1, is located in its N-terminal arm (NTA). The NTA establishes an extensive network of interactions on the inner capsid surface that stabilizes the hexamers and pentamers. For this structural polymorphism, we show that the NTA must interact with the RNA viral genome, that is, the genomic RNA acts with the NTA as a molecular co-switch. The NTA-RNA interaction leads to specific conformational states that result in two types of VP1 dimers (the basic building blocks) necessary for T = 3 capsid assembly. In addition, we used atomic force microscopy (AFM) to assess whether differences in genomic RNA content influence viral properties such as capsid stiffness in physiological conditions. These analyses highlight the mechanical role of packed RNA genome in RHDV virions, as the virion capsid pentamers are strengthened by interactions of the NTA star-like structure promoted by the viral genome. These results indicate that the interactions between the NTA and the viral genome guide the conformational states of VP1 dimers, directing capsid assembly and modulating its mechanical properties. Through interference with intermediate assemblies, the NTA network promoted by the genome could be an attractive target in future antiviral strategies.
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Dec 2025
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Krios I-Titan Krios I at Diamond
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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.
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Dec 2025
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