Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[23268]
Open Access
Abstract: Most eukaryotic messenger RNAs (mRNAs) are processed at their 3′ end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3′ end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3′ end processing, activating CPF, and ensuring timely transcription termination.
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May 2022
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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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Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Soledad
Stagnoli
,
Francesca
Peccati
,
Sean R.
Connell
,
Ane
Martinez-Castillo
,
Diego
Charro
,
Oscar
Millet
,
Chiara
Bruzzone
,
Asis
Palazon
,
Ana
Ardá
,
Jesús
Jiménez-Barbero
,
June
Ereño-Orbea
,
Nicola G. A.
Abrescia
,
Gonzalo
Jiménez-Osés
Diamond Proposal Number(s):
[23872]
Open Access
Abstract: Two years after its emergence, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several vaccines. The extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin converting enzyme 2 (ACE2) through its receptor binding domain (RBD), is the major target of neutralizing antibodies. Like to many other viral fusion proteins, the SARS-CoV-2 spike protein utilizes a glycan shield to thwart the host immune response. To grasp the influence of chemical signatures on carbohydrate mobility and reconcile the cryo-EM density of specific glycans we combined our cryo-EM map of the S ectodomain to 4.1 Å resolution, reconstructed from a limited number of particles, and all-atom molecular dynamics simulations. Chemical modifications modeled on representative glycans (defucosylation, sialylation and addition of terminal LacNAc units) show no significant influence on either protein shielding or glycan flexibility. By estimating at selected sites the local correlation between the full density map and atomic model-based maps derived from molecular dynamics simulations, we provide insight into the geometries of the α-Man-(1→3)-[α-Man-(1→6)-]-β-Man-(1→4)-β-GlcNAc(1→4)-β-GlcNAc core common to all N-glycosylation sites.
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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 II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[26464, 28151]
Open Access
Abstract: Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that by collecting cryo-ET STA data using the same conditions as SPA, with both Correlated Double Sampling (CDS) and super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising data size on disk and area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).
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Apr 2022
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Krios III-Titan Krios III at Diamond
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Manuel
Schweighauser
,
Diana
Arseni
,
Mehtap
Bacioglu
,
Melissa
Huang
,
Sofia
Lovestam
,
Yang
Shi
,
Yang
Yang
,
Wenjuan
Zhang
,
Abhay
Kotecha
,
Holly J.
Garringer
,
Ruben
Vidal
,
Grace I.
Hallin
,
Kathy L.
Newell
,
Airi
Tarutani
,
Shigeo
Murayama
,
Masayuki
Miyazaki
,
Yuko
Saito
,
Mari
Yoshida
,
Kazuko
Hasegawa
,
Tammaryn
Lashley
,
Tamas
Revesz
,
Gabor G.
Kovacs
,
John
Van Swieten
,
Masaki
Takao
,
Masato
Hasegawa
,
Bernardino
Ghetti
,
Maria Grazia
Spillantini
,
Benjamin
Ryskeldi-Falcon
,
Alexey G.
Murzin
,
Michel
Goedert
,
Sjors H. W.
Scheres
Diamond Proposal Number(s):
[17434, 23268]
Abstract: Many age-dependent neurodegenerative diseases, like Alzheimer’s and Parkinson’s, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-β (Aβ), α-synuclein and TDP-43 are the most common1,2. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the cryo-EM structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including sporadic and inherited tauopathies, Aβ-amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 neurologically normal individuals with no or only few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29 kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific for the C-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.
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Mar 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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