Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[22006, 30374]
Open Access
Abstract: During synthesis of the ribosomal RNA precursor, RNA polymerase I (Pol I) monitors DNA integrity but its response to DNA damage remains poorly studied. Abasic sites are among the most prevalent DNA lesions in eukaryotic cells, and their detection is critical for cell survival. We report cryo-EM structures of Pol I in different stages of stalling at abasic sites, supported by in vitro transcription studies. Slow nucleotide addition opposite abasic sites occurs through base sandwiching between the RNA 3′-end and the Pol I bridge helix. Templating abasic sites can also cause Pol I cleft opening, which enables the A12 subunit to access the active center. Nucleotide addition opposite the lesion induces a translocation intermediate where DNA bases tilt to form hydrogen bonds with the new RNA base. These findings reveal unique mechanisms of Pol I stalling at abasic sites, differing from arrest by bulky lesions or abasic site handling by RNA polymerase II.
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Jun 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[28713]
Open Access
Abstract: Muscle-type nicotinic acetylcholine receptor (AChR) is the key signaling molecule in neuromuscular junctions. Here, we present the structures of full-length human adult receptors in complex with Fab35 in α-bungarotoxin (αBuTx)-bound resting states and ACh-bound desensitized states. In addition to identifying the conformational changes during recovery from desensitization, we also used electrophysiology to probe the effects of eight previously unstudied AChR genetic variants found in patients with congenital myasthenic syndrome (CMS), revealing they cause either slow- or fast-channel CMS characterized by prolonged or abbreviated ion channel bursts. The combined kinetic and structural data offer a better understanding of both the AChR state transition and the pathogenic mechanisms of disease variants.
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May 2025
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Krios I-Titan Krios I at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[20287, 26703]
Open Access
Abstract: Bacterial mating, or conjugation, was discovered nearly 80 years ago as a process transferring genes from one bacterial cell (the donor) to another (the recipient). It requires three key multiprotein complexes in the donor cell: a DNA-processing machinery called the relaxosome, a double-membrane spanning type 4 secretion system (T4SS), and an extracellular appendage termed pilus. While the near-atomic resolution structures of the T4SS and pilus are already known, that of the relaxosome has not been reported to date. Here, we describe the cryo-EM structure of the fully assembled relaxosome encoded by the paradigm F plasmid in two different states corresponding to distinct functional steps along the DNA processing reaction. By varying the structures of model DNAs we delineate conformational changes required to initiate conjugation. Mutational studies of the various protein-protein and protein-DNA interaction hubs suggest a complex sensitive to trigger signals, that could arise from cell-to-cell contacts with recipient cells.
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May 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[29812]
Open Access
Abstract: During HIV-1 maturation, the matrix (MA) lattice underlying the viral membrane undergoes a structural rearrangement, and the newly released capsid (CA) protein forms a mature CA. While it is well established that CA formation is essential for particle infectivity, the functional role of MA structural maturation remains unclear. Here, we examine maturation of an MA triple mutant, L20K/E73K/A82T, which, despite replicating similarly to wild-type (WT) in some cell lines, exhibits distinct biochemical behaviors that suggest altered MA-MA interactions. Cryo–electron tomography with subtomogram averaging reveals that, although the MA lattice in immature L20K/E73K/A82T virions closely resembles that of the WT, mature L20K/E73K/A82T virions lack a detectable MA lattice. All-atom molecular dynamics simulations suggest that this absence results from destabilized inter-trimer MA interactions in mature L20K/E73K/A82T mutant virions. These findings suggest that an ordered, membrane-associated mature MA lattice is not essential for HIV-1 infectivity, providing insights into the structural requirements for HIV-1 particle maturation and generation of infectious particles.
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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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Krios I-Titan Krios I at Diamond
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Tarik
Husremović
,
Vanessa
Meier
,
Lucas
Piëch
,
Katharina M.
Siess
,
Sumire
Antonioli
,
Irina
Grishkovskaya
,
Nikoleta
Kircheva
,
Silvia E.
Angelova
,
Karoline
Wenzl
,
Andreas
Brandstätter
,
Jiri
Veis
,
Fran
Miočić-Stošić
,
Dorothea
Anrather
,
Markus
Hartl
,
Linda
Truebestein
,
Luis M.
Cerron-Alvan
,
Martin
Leeb
,
Bojan
Žagrović
,
Stephan
Hann
,
Christoph
Bock
,
Egon
Ogris
,
Todor
Dudev
,
Nicholas A. T.
Irwin
,
David
Haselbach
,
Thomas A.
Leonard
Diamond Proposal Number(s):
[25222]
Open Access
Abstract: The phosphoinositide 3-kinase (PI3K) pathway is a major regulator of cell and organismal growth. Consequently, hyperactivation of PI3K and its downstream effector kinase, Akt, is observed in many human cancers. Pleckstrin homology domain leucine-rich repeat-containing protein phosphatases (PHLPP), two paralogous members of the metal-dependent protein phosphatase family, have been reported as negative regulators of Akt signaling and, therefore, tumor suppressors. However, the stoichiometry and identity of the bound metal ion(s), mechanism of action, and enzymatic specificity of these proteins are not known. Seeking to fill these gaps in our understanding of PHLPP biology, we unexpectedly found that PHLPP2 has no catalytic activity in vitro. Instead, we found that PHLPP2 is a pseudophosphatase with a single zinc ion bound in its catalytic center. Furthermore, we found that cancer genomics data do not support the proposed role of PHLPP1 or PHLPP2 as tumor suppressors. Phylogenetic analyses revealed an ancestral phosphatase that arose more than 1,000 Mya, but that lost activity at the base of the metazoan lineage. Surface conservation indicates that while PHLPP2 has lost catalytic activity, it may have retained substrate binding. Finally, using phylogenomics, we identify coevolving genes consistent with a scaffolding role for PHLPP2 on membranes. In summary, our results provide a molecular explanation for the inconclusive results that have hampered research on PHLPP and argue for a focus on the noncatalytic roles of PHLPP1 and PHLPP2.
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Apr 2025
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Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Nattapong
Sanguankiattichai
,
Balakumaran
Chandrasekar
,
Yuewen
Sheng
,
Nathan
Hardenbrook
,
Werner W. A.
Tabak
,
Margit
Drapal
,
Farnusch
Kaschani
,
Clemens
Grünwald-Gruber
,
Daniel
Krahn
,
Pierre
Buscaill
,
Suzuka
Yamamoto
,
Atsushi
Kato
,
Robert
Nash
,
George
Fleet
,
Richard
Strasser
,
Paul D.
Fraser
,
Markus
Kaiser
,
Peijun
Zhang
,
Gail M.
Preston
,
Renier A. L.
Van Der Hoorn
Diamond Proposal Number(s):
[21004, 29812, 28713]
Abstract: The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo–electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
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Apr 2025
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[31586]
Abstract: Human H ferritin (HuHf) has excellent potential as a nanocarrier for the selective delivery of anticancer metal-based drugs to tumour cells. Here, we addressed the interaction of the gold monocarbene compound Au(NHC)Cl with HuHf by electrospray ionization-mass spectrometry (ESI-MS) measurements, which provide the metalation state of the protein subunits and demonstrate the involvement of protein cysteines in gold binding. The adduct between Au(NHC)Cl and HuHf was studied by cryo-EM measurements, resulting in a high-resolution 3D density map at 1.51 Å. The cryo-EM structure shows a novel tetranuclear gold(I) cluster, located in a surface pocket of each subunit where it is bound to Cys90 and Cys102. The short inter-metal distances are diagnostic of the occurrence of aurophilic interactions. The present work demonstrates the usefulness of cryo-EM to investigate the interactions between metal-based drugs and their protein targets/carriers also leveraging the strong signal of transition metal ions.
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Apr 2025
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[21404]
Open Access
Abstract: Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.
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Apr 2025
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[31336]
Open Access
Abstract: Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates ‘eat me’ signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4–RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.
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Apr 2025
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