Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[2154]
Abstract: Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo–electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.
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May 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Rungtiwa
Nutalai
,
Daming
Zhou
,
Aekkachai
Tuekprakhon
,
Helen M.
Ginn
,
Piyada
Supasa
,
Chang
Liu
,
Jiandong
Huo
,
Alexander J.
Mentzer
,
Helen M. E.
Duyvesteyn
,
Aiste
Dijokaite-Guraliuc
,
Donal
Skelly
,
Thomas G.
Ritter
,
Ali
Amini
,
Sagida
Bibi
,
Sandra
Adele
,
Sile Ann
Johnson
,
Bede
Constantinides
,
Hermione
Webster
,
Nigel
Temperton
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Derrick
Crook
,
Andrew J.
Pollard
,
Teresa
Lambe
,
Philip
Goulder
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Juthathip
Mongkolsapaya
,
Elizabeth E.
Fry
,
Wanwisa
Dejnirattisai
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
,
Christopher
Conlon
,
Alexandra
Deeks
,
John
Frater
,
Lisa
Frending
,
Siobhan
Gardiner
,
Anni
Jämsén
,
Katie
Jeffery
,
Tom
Malone
,
Eloise
Phillips
,
Lucy
Rothwell
,
Lizzie
Stafford
Diamond Proposal Number(s):
[27009, 26983]
Open Access
Abstract: Highly transmissible Omicron variants of SARS-CoV-2 currently dominate globally. Here, we compare neutralization of Omicron BA.1, BA.1.1 and BA.2. BA.2 RBD has slightly higher ACE2 affinity than BA.1 and slightly reduced neutralization by vaccine serum, possibly associated with its increased transmissibility. Neutralization differences between sub-lineages for mAbs (including therapeutics) mostly arise from variation in residues bordering the ACE2 binding site, however, more distant mutations S371F (BA.2) and R346K (BA.1.1) markedly reduce neutralization by therapeutic antibody Vir-S309. In-depth structure-and-function analyses of 27 potent RBD-binding mAbs isolated from vaccinated volunteers following breakthrough Omicron-BA.1 infection reveals that they are focussed in two main clusters within the RBD, with potent right-shoulder antibodies showing increased prevalence. Selection and somatic maturation have optimized antibody potency in less-mutated epitopes and recovered potency in highly mutated epitopes. All 27 mAbs potently neutralize early pandemic strains and many show broad reactivity with variants of concern.
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May 2022
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B24-Cryo Soft X-ray Tomography
I13-2-Diamond Manchester Imaging
Krios I-Titan Krios I at Diamond
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Open Access
Abstract: As sample preparation and imaging techniques have expanded and improved to include a variety of options for larger sized and numbers of samples, the bottleneck in volumetric imaging is now data analysis. Annotation and segmentation are both common, yet difficult, data analysis tasks which are required to bring meaning to the volumetric data. The SuRVoS application has been updated and redesigned to provide access to both manual and machine learning-based segmentation and annotation techniques, including support for crowd sourced data. Combining adjacent, similar voxels (supervoxels) provides a mechanism for speeding up segmentation both in the painting of annotation and by training a segmentation model on a small amount of annotation. The support for layers allows multiple datasets to be viewed and annotated together which, for example, enables the use of correlative data (e.g. crowd-sourced annotations or secondary imaging techniques) to guide segmentation. The ability to work with larger data on high-performance servers with GPUs has been added through a client-server architecture and the Pytorch-based image processing and segmentation server is flexible and extensible, and allows the implementation of deep learning-based segmentation modules. The client side has been built around Napari allowing integration of SuRVoS into an ecosystem for open-source image analysis while the server side has been built with cloud computing and extensibility through plugins in mind. Together these improvements to SuRVoS provide a platform for accelerating the annotation and segmentation of volumetric and correlative imaging data across modalities and scales.
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Apr 2022
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[23297]
Open Access
Abstract: In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes and ordered chromatin loops. It remains unknown if these cells have any other large, meiosis-related intranuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no cryo-ET densities that resemble dense ladder-like structures or ordered chromatin loops. Instead, we found large numbers of 12-nm-wide triple-helices that pack into ordered bundles. These structures, herein called meiotic triple helices (MTHs), are present in meiotic cells, but not in interphase cells. MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same timeframe as synaptonemal complexes (SCs) in wild-type cells and in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs coexist with previously unreported large, ordered assemblies.
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Apr 2022
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Krios I-Titan Krios I at Diamond
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Abstract: Kinetochores assemble onto specialized centromeric CENP-A nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-EM structures of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the α-satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.
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Apr 2022
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Krios I-Titan Krios I at Diamond
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John J.
Kelly
,
Dale
Tranter
,
Els
Pardon
,
Gamma
Chi
,
Holger
Kramer
,
Lotta
Happonen
,
Kelly M.
Knee
,
Jay M.
Janz
,
Jan
Steyaert
,
Christine
Bulawa
,
Ville O.
Paavilainen
,
Juha T.
Huiskonen
,
Wyatt W.
Yue
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.
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Apr 2022
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[22238]
Open Access
Abstract: Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1—both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome — license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.
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Mar 2022
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Krios I-Titan Krios I at Diamond
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Pu
Qian
,
Alastair T.
Gardiner
,
Ivana
Šímová
,
Katerina
Naydenova
,
Tristan I.
Croll
,
Philip J.
Jackson
,
Nupur
Nupur
,
Miroslav
Kloz
,
Petra
Čubáková
,
Marek
Kuzma
,
Yonghui
Zeng
,
Pablo
Castro-Hartmann
,
Bart
Van Knippenberg
,
Kenneth N.
Goldie
,
David
Kaftan
,
Pavel
Hrouzek
,
Jan
Hájek
,
Jon
Agirre
,
C. Alistair
Siebert
,
David
Bína
,
Kasim
Sader
,
Henning
Stahlberg
,
Roman
Sobotka
,
Christopher J.
Russo
,
Tomáš
Polívka
,
C. Neil
Hunter
,
Michal
Koblížek
Diamond Proposal Number(s):
[29785]
Open Access
Abstract: Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.
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Feb 2022
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Krios I-Titan Krios I at Diamond
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Maria Teresa
Bueno-Carrasco
,
Jorge
Cuellar
,
Marte I.
Flydal
,
César
Santiago
,
Trond-André
Kråkenes
,
Rune
Kleppe
,
José R.
López-Blanco
,
Miguel
Marcilla
,
Knut
Teigen
,
Sara
Alvira
,
Pablo
Chacón
,
Aurora
Martinez
,
José Maria
Valpuesta
Diamond Proposal Number(s):
[15997, 22006]
Open Access
Abstract: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.
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Jan 2022
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Krios I-Titan Krios I at Diamond
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Jennifer
Ross
,
Zak
Mciver
,
Thomas
Lambert
,
Cecilia
Piergentili
,
Jasmine Emma
Bird
,
Kelly J.
Gallagher
,
Faye L.
Cruickshank
,
Patrick
James
,
Efrain
Zarazúa-Arvizu
,
Louise E.
Horsfall
,
Kevin J.
Waldron
,
Marcus D.
Wilson
,
C. Logan
Mackay
,
Arnaud
Basle
,
David J.
Clarke
,
Jon
Marles-Wright
Diamond Proposal Number(s):
[16637]
Open Access
Abstract: Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo–electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.
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Jan 2022
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