Krios III-Titan Krios III at Diamond
|
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: We used electron cryo-microscopy (cryo-EM) to determine the structures of Aβ40 filaments from the leptomeninges of individuals with Alzheimer’s disease and cerebral amyloid angiopathy. In agreement with previously reported structures, which were solved to a resolution of 4.4 Å, we found three types of filaments. However, our new structures, solved to a resolution of 2.4 Å, revealed differences in the sequence assignment that redefine the fold of Aβ40 peptides and their interactions. Filaments are made of pairs of protofilaments, the ordered core of which comprises D1–G38. The different filament types comprise one, two or three protofilament pairs. In each pair, residues H14–G37 of both protofilaments adopt an extended conformation and pack against each other in an anti-parallel fashion, held together by hydrophobic interactions and hydrogen bonds between main chains and side chains. Residues D1–H13 fold back on the adjacent parts of their own chains through both polar and non-polar interactions. There are also several additional densities of unknown identity. Sarkosyl extraction and aqueous extraction gave the same structures. By cryo-EM, parenchymal deposits of Aβ42 and blood vessel deposits of Aβ40 have distinct structures, supporting the view that Alzheimer’s disease and cerebral amyloid angiopathy are different Aβ proteinopathies.
|
Dec 2023
|
|
Krios I-Titan Krios I at Diamond
Krios III-Titan Krios III at Diamond
|
Diamond Proposal Number(s):
[31336]
Open Access
Abstract: The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo–electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-ANuc). This revealed a central CENP-ANuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-ANuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.
|
Jul 2023
|
|
Krios IV-Titan Krios IV at Diamond
|
Yang
Yang
,
Holly J.
Garringer
,
Yang
Shi
,
Sofia
Lovestam
,
Sew
Peak-Chew
,
Xianjun
Zhang
,
Abhay
Kotecha
,
Mehtap
Bacioglu
,
Atsuo
Koto
,
Masaki
Takao
,
Maria Grazia
Spillantini
,
Bernardino
Ghetti
,
Ruben
Vidal
,
Alexey G.
Murzin
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36–100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.
|
Feb 2023
|
|
Krios I-Titan Krios I at Diamond
|
Yang
Yang
,
Wenjuan
Zhang
,
Alexey G.
Murzin
,
Manuel
Schweighauser
,
Melissa
Huang
,
Sofia
Lovestam
,
Sew Y.
Peak-Chew
,
Takashi
Saito
,
Takaomi C.
Saido
,
Jennifer
Macdonald
,
Isabelle
Lavenir
,
Bernardino
Ghetti
,
Caroline
Graff
,
Amit
Kumar
,
Agneta
Nordberg
,
Michel
Goedert
,
Sjors H. W.
Scheres
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer’s disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12–V40 (human Arctic fold A) and E11–G37 (human Arctic fold B). They have a substructure (residues F20–G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line AppNL−G−F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL−G−F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL−G−F murine Arctic fold differs from the human Arctic folds, but shares some substructure.
|
Jan 2023
|
|
Krios IV-Titan Krios IV at Diamond
|
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.
|
Sep 2022
|
|
Krios III-Titan Krios III at Diamond
|
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.
|
Mar 2022
|
|
Krios I-Titan Krios I at Diamond
|
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.
|
Mar 2021
|
|
Krios I-Titan Krios I at Diamond
|
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.
|
Sep 2020
|
|
Krios I-Titan Krios I at Diamond
|
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.
|
Nov 2019
|
|
Krios III-Titan Krios III at Diamond
|
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.
|
Jul 2019
|
|