Krios IV-Titan Krios IV at Diamond
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Yang
Yang
,
Yang
Shi
,
Manuel
Schweighauser
,
Xianjun
Zhang
,
Abhay
Kotecha
,
Alexey G.
Murzin
,
Holly J.
Garringer
,
Patrick W.
Cullinane
,
Yuko
Saito
,
Tatiana
Foroud
,
Thomas T.
Warner
,
Kazuko
Hasegawa
,
Ruben
Vidal
,
Shigeo
Murayama
,
Tamas
Revesz
,
Bernardino
Ghetti
,
Masato
Hasegawa
,
Tammaryn
Lashley
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[23268]
Abstract: Parkinson’s disease (PD) is the most common movement disorder, with resting tremor, rigidity, bradykinesia and postural instability being major symptoms1. Neuropathologically, it is characterized by the presence of abundant filamentous inclusions of α-synuclein in the form of Lewy bodies and Lewy neurites in some brain cells, including dopaminergic nerve cells of the substantia nigra2. PD is increasingly recognised as a multisystem disorder, with cognitive decline being one of its most common non-motor symptoms. Many patients with PD develop dementia more than 10 years after diagnosis3. PD dementia (PDD) is clinically and neuropathologically similar to dementia with Lewy bodies (DLB), which is diagnosed when cognitive impairment precedes parkinsonian motor signs or begins within one year from their onset4. In PDD, cognitive impairment develops in the setting of well-established PD. Besides PD and DLB, multiple system atrophy (MSA) is the third major synucleinopathy5. It is characterized by the presence of abundant filamentous α-synuclein inclusions in brain cells, especially oligodendrocytes (Papp-Lantos bodies). We previously reported the electron cryo-microscopy structures of two types of α-synuclein filament extracted from the brains of individuals with MSA6. Each filament type is made of two different protofilaments. Here we report that the cryo-electron microscopy structures of α-synuclein filaments from the brains of individuals with PD, PDD and DLB are made of a single protofilament (Lewy fold) that is markedly different from the protofilaments of MSA. These findings establish the existence of distinct molecular conformers of assembled α-synuclein in neurodegenerative disease.
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Sep 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]
Open Access
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
Krios II-Titan Krios II at Diamond
Krios III-Titan Krios III at Diamond
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Luiza
Mendonca
,
Dapeng
Sun
,
Jiying
Ning
,
Jiwei
Liu
,
Abhay
Kotecha
,
Mateusz
Olek
,
Thomas
Frosio
,
Xiaofeng
Fu
,
Benjamin A.
Himes
,
Alex B.
Kleinpeter
,
Eric O.
Freed
,
Jing
Zhou
,
Christopher
Aiken
,
Peijun
Zhang
Diamond Proposal Number(s):
[18477, 21005, 21004]
Open Access
Abstract: Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.
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Apr 2021
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Krios IV-Titan Krios IV at Diamond
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Max
Renner
,
Wanwisa
Dejnirattisai
,
Loic
Carrique
,
Itziar
Serna Martin
,
Dimple
Karia
,
Serban L.
Ilca
,
Shu F.
Ho
,
Abhay
Kotecha
,
Jeremy R.
Keown
,
Juthathip
Mongkolsapaya
,
Gavin R.
Screaton
,
Jonathan M.
Grimes
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.
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Feb 2021
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I03-Macromolecular Crystallography
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Kuan-Ying A.
Huang
,
Daming
Zhou
,
Elizabeth E.
Fry
,
Abhay
Kotecha
,
Peng-Nien
Huang
,
Shu-Li
Yang
,
Kuo-Chien
Tsao
,
Yhu-Chering
Huang
,
Tzou-Yien
Lin
,
Jingshan
Ren
,
David I.
Stuart
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.
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Oct 2020
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[18477]
Open Access
Abstract: Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.
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Sep 2020
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Yuguang
Zhao
,
Daming
Zhou
,
Tao
Ni
,
Dimple
Karia
,
Abhay
Kotecha
,
Xiangxi
Wang
,
Zihe
Rao
,
E. Yvonne
Jones
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
David I.
Stuart
Open Access
Abstract: Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.
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Jan 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Jingshan
Ren
,
Joanne E.
Nettleship
,
Gemma
Harris
,
William
Mwangi
,
Nahid
Rhaman
,
Clare
Grant
,
Abhay
Kotecha
,
Elizabeth
Fry
,
Bryan
Charleston
,
David I.
Stuart
,
John
Hammond
,
Raymond J.
Owens
Diamond Proposal Number(s):
[10627, 14744]
Open Access
Abstract: Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.
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Aug 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[14856]
Abstract: Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1,2,3,4,5,6,7,8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10,11,12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10–12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11,12,13,14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.
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May 2019
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Kamel
El Omari
,
Sai
Li
,
Abhay
Kotecha
,
Thomas S.
Walter
,
Eduardo A.
Bignon
,
Karl
Harlos
,
Pentti
Somerharju
,
Felix
De Haas
,
Daniel K.
Clare
,
Mika
Molin
,
Felipe
Hurtado
,
Mengqiu
Li
,
Jonathan
Grimes
,
Dennis H.
Bamford
,
Nicole D.
Tischler
,
Juha T.
Huiskonen
,
Dave I.
Stuart
,
Elina
Roine
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.
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Feb 2019
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