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]
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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Yang
Shi
,
Alexey G.
Murzin
,
Benjamin
Falcon
,
Alexander
Epstein
,
Jonathan
Machin
,
Paul
Tempest
,
Kathy L.
Newell
,
Ruben
Vidal
,
Holly J.
Garringer
,
Naruhiko
Sahara
,
Makoto
Higuchi
,
Bernardino
Ghetti
,
Ming-Kuei
Jang
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Tau and Aβ assemblies of Alzheimer’s disease (AD) can be visualized in living subjects using positron emission tomography (PET). Tau assemblies comprise paired helical and straight filaments (PHFs and SFs). APN-1607 (PM-PBB3) is a recently described PET ligand for AD and other tau proteinopathies. Since it is not known where in the tau folds PET ligands bind, we used electron cryo-microscopy (cryo-EM) to determine the binding sites of APN-1607 in the Alzheimer fold. We identified two major sites in the β-helix of PHFs and SFs and a third major site in the C-shaped cavity of SFs. In addition, we report that tau filaments from posterior cortical atrophy (PCA) and primary age-related tauopathy (PART) are identical to those from AD. In support, fluorescence labelling showed binding of APN-1607 to intraneuronal inclusions in AD, PART and PCA. Knowledge of the binding modes of APN-1607 to tau filaments may lead to the development of new ligands with increased specificity and binding activity. We show that cryo-EM can be used to identify the binding sites of small molecules in amyloid filaments.
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Mar 2021
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Krios I-Titan Krios I at Diamond
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Manuel
Schweighauser
,
Yang
Shi
,
Airi
Tarutani
,
Fuyuki
Kametani
,
Alexey G.
Murzin
,
Bernardino
Ghetti
,
Tomoyasu
Matsubara
,
Taisuke
Tomita
,
Takashi
Ando
,
Kazuko
Hasegawa
,
Shigeo
Murayama
,
Mari
Yoshida
,
Masato
Hasegawa
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[17434]
Abstract: Synucleinopathies, which include multiple system atrophy (MSA), Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies (DLB), are human neurodegenerative diseases. Existing treatments are at best symptomatic. These diseases are characterized by the presence of, and believed to be caused by the formation of, filamentous inclusions of α-synuclein in brain cells2,3. However, the structures of α-synuclein filaments from the human brain are unknown. Here, using cryo-electron microscopy, we show that α-synuclein inclusions from the brains of individuals with MSA are made of two types of filament, each of which consists of two different protofilaments. In each type of filament, non-proteinaceous molecules are present at the interface of the two protofilaments. Using two-dimensional class averaging, we show that α-synuclein filaments from the brains of individuals with MSA differ from those of individuals with DLB, which suggests that distinct conformers or strains characterize specific synucleinopathies. As is the case with tau assemblies4,5,6,7,8,9, the structures of α-synuclein filaments extracted from the brains of individuals with MSA differ from those formed in vitro using recombinant proteins, which has implications for understanding the mechanisms of aggregate propagation and neurodegeneration in the human brain. These findings have diagnostic and potential therapeutic relevance, especially because of the unmet clinical need to be able to image filamentous α-synuclein inclusions in the human brain.
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Sep 2020
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Krios I-Titan Krios I at Diamond
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Shabih
Shakeel
,
Eeson
Rajendra
,
Pablo
Alcon
,
Francis
O'Reilly
,
Dror S.
Chorev
,
Sarah
Maslen
,
Gianluca
Degliesposti
,
Christopher J.
Russo
,
Shaoda
He
,
Chris H.
Hill
,
J. Mark
Skehel
,
Sjors H. W.
Scheres
,
Ketan J.
Patel
,
Juri
Rappsilber
,
Carol V.
Robinson
,
Lori A.
Passmore
Diamond Proposal Number(s):
[18091, 17434]
Abstract: The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2–FANCI4,5 by the FA core complex—a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.
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Nov 2019
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.
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Jul 2019
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Krios I-Titan Krios I at Diamond
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Benjamin
Falcon
,
Jasenko
Zivanov
,
Wenjuan
Zhang
,
Alexey G.
Murzin
,
Holly J.
Garringer
,
Ruben
Vidal
,
R. Anthony
Crowther
,
Kathy L.
Newell
,
Bernardino
Ghetti
,
Michel
Goedert
,
Sjors H. W.
Scheres
Diamond Proposal Number(s):
[17434]
Abstract: Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy that is associated with repetitive head impacts or exposure to blast waves. First described as punch-drunk syndrome and dementia pugilistica in retired boxers1,2,3, CTE has since been identified in former participants of other contact sports, ex-military personnel and after physical abuse4,5,6,7. No disease-modifying therapies currently exist, and diagnosis requires an autopsy. CTE is defined by an abundance of hyperphosphorylated tau protein in neurons, astrocytes and cell processes around blood vessels8,9. This, together with the accumulation of tau inclusions in cortical layers II and III, distinguishes CTE from Alzheimer’s disease and other tauopathies10,11. However, the morphologies of tau filaments in CTE and the mechanisms by which brain trauma can lead to their formation are unknown. Here we determine the structures of tau filaments from the brains of three individuals with CTE at resolutions down to 2.3 Å, using cryo-electron microscopy. We show that filament structures are identical in the three cases but are distinct from those of Alzheimer’s and Pick’s diseases, and from those formed in vitro12,13,14,15. Similar to Alzheimer’s disease12,14,16,17,18, all six brain tau isoforms assemble into filaments in CTE, and residues K274–R379 of three-repeat tau and S305–R379 of four-repeat tau form the ordered core of two identical C-shaped protofilaments. However, a different conformation of the β-helix region creates a hydrophobic cavity that is absent in tau filaments from the brains of patients with Alzheimer’s disease. This cavity encloses an additional density that is not connected to tau, which suggests that the incorporation of cofactors may have a role in tau aggregation in CTE. Moreover, filaments in CTE have distinct protofilament interfaces to those of Alzheimer’s disease. Our structures provide a unifying neuropathological criterion for CTE, and support the hypothesis that the formation and propagation of distinct conformers of assembled tau underlie different neurodegenerative diseases.
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Mar 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Assembly of microtubule-associated protein tau into filamentous inclusions underlies a range of neurodegenerative diseases. Tau filaments adopt different conformations in Alzheimer’s and Pick’s diseases. Here, we used cryo- and immuno- electron microscopy to characterise filaments that were assembled from recombinant full-length human tau with four (2N4R) or three (2N3R) microtubule-binding repeats in the presence of heparin. 2N4R tau assembles into multiple types of filaments, and the structures of three types reveal similar ‘kinked hairpin’ folds, in which the second and third repeats pack against each other. 2N3R tau filaments are structurally homogeneous, and adopt a dimeric core, where the third repeats of two tau molecules pack in a parallel manner. The heparin-induced tau filaments differ from those of Alzheimer’s or Pick’s disease, which have larger cores with different repeat compositions. Our results illustrate the structural versatility of amyloid filaments, and raise questions about the relevance of in vitro assembly.
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Feb 2019
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[17434]
Open Access
Abstract: The ordered assembly of tau protein into abnormal filaments is a defining characteristic of Alzheimer’s disease (AD) and other neurodegenerative disorders. It is not known if the structures of tau filaments vary within, or between, the brains of individuals with AD. We used a combination of electron cryo-microscopy (cryo-EM) and immuno-gold negative-stain electron microscopy (immuno-EM) to determine the structures of paired helical filaments (PHFs) and straight filaments (SFs) from the frontal cortex of 17 cases of AD (15 sporadic and 2 inherited) and 2 cases of atypical AD (posterior cortical atrophy). The high-resolution structures of PHFs and SFs from the frontal cortex of 3 cases of AD, 2 sporadic and 1 inherited, were determined by cryo-EM. We also used immuno-EM to study the PHFs and SFs from a number of cortical and subcortical brain regions. PHFs outnumbered SFs in all AD cases. By cryo-EM, PHFs and SFs were made of two C-shaped protofilaments with a combined cross-β/β-helix structure, as described previously for one case of AD. The higher resolution structures obtained here showed two additional amino acids at each end of the protofilament. The immuno-EM findings, which indicated the presence of repeats 3 and 4, but not of the N-terminal regions of repeats 1 and 2, of tau in the filament cores of all AD cases, were consistent with the cryo-EM results. These findings show that there is no significant variation in tau filament structures between individuals with AD. This knowledge will be crucial for understanding the mechanisms that underlie tau filament formation and for developing novel diagnostics and therapies.
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Nov 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13212]
Open Access
Abstract: Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.
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Jan 2018
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