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- PDB-8ceb: Type2 alpha-synuclein filament assembled in vitro by wild-type an... -

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Basic information

Entry
Database: PDB / ID: 8ceb
TitleType2 alpha-synuclein filament assembled in vitro by wild-type and mutant (7 residues insertion) protein
ComponentsAlpha-synuclein
KeywordsPROTEIN FIBRIL / alpha-synuclein / amyloid / fibril / insertion / mutation / in vitro / recombinant / synucleinopathy
Function / homology
Function and homology information


negative regulation of mitochondrial electron transport, NADH to ubiquinone / neutral lipid metabolic process / regulation of phospholipase activity / negative regulation of monooxygenase activity / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of glutathione peroxidase activity / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process ...negative regulation of mitochondrial electron transport, NADH to ubiquinone / neutral lipid metabolic process / regulation of phospholipase activity / negative regulation of monooxygenase activity / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of glutathione peroxidase activity / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / negative regulation of transporter activity / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / positive regulation of protein localization to cell periphery / regulation of synaptic vesicle recycling / negative regulation of platelet-derived growth factor receptor signaling pathway / negative regulation of exocytosis / regulation of glutamate secretion / response to iron(II) ion / regulation of norepinephrine uptake / dopamine biosynthetic process / SNARE complex assembly / positive regulation of neurotransmitter secretion / regulation of locomotion / synaptic vesicle priming / positive regulation of inositol phosphate biosynthetic process / regulation of macrophage activation / mitochondrial ATP synthesis coupled electron transport / dopamine uptake involved in synaptic transmission / negative regulation of microtubule polymerization / synaptic vesicle transport / dynein complex binding / positive regulation of receptor recycling / regulation of dopamine secretion / protein kinase inhibitor activity / negative regulation of thrombin-activated receptor signaling pathway / response to type II interferon / cuprous ion binding / positive regulation of exocytosis / synaptic vesicle exocytosis / positive regulation of endocytosis / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / response to magnesium ion / kinesin binding / regulation of presynapse assembly / synaptic vesicle endocytosis / alpha-tubulin binding / negative regulation of serotonin uptake / localization / phospholipid metabolic process / axon terminus / supramolecular fiber organization / inclusion body / cellular response to copper ion / Hsp70 protein binding / cellular response to epinephrine stimulus / excitatory postsynaptic potential / response to interleukin-1 / adult locomotory behavior / SNARE binding / positive regulation of release of sequestered calcium ion into cytosol / fatty acid metabolic process / long-term synaptic potentiation / regulation of transmembrane transporter activity / ferrous iron binding / protein tetramerization / phosphoprotein binding / synapse organization / regulation of long-term neuronal synaptic plasticity / microglial cell activation / negative regulation of protein kinase activity / phospholipid binding / protein destabilization / PKR-mediated signaling / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / tau protein binding / positive regulation of protein serine/threonine kinase activity / receptor internalization / synaptic vesicle membrane / positive regulation of inflammatory response / activation of cysteine-type endopeptidase activity involved in apoptotic process / actin cytoskeleton / positive regulation of peptidyl-serine phosphorylation / cellular response to oxidative stress / cell cortex / actin binding / histone binding / growth cone / chemical synaptic transmission / postsynapse / neuron apoptotic process / negative regulation of neuron apoptotic process / amyloid fibril formation / response to lipopolysaccharide / lysosome / oxidoreductase activity / molecular adaptor activity / transcription cis-regulatory region binding
Similarity search - Function
Synuclein / Alpha-synuclein / Synuclein
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsYang, Y. / Garringer, J.H. / Shi, Y. / Lovestam, S. / Sew, P.C. / Zhang, X.J. / Kotecha, A. / Bacioglu, M. / Koto, A. / Takao, M. ...Yang, Y. / Garringer, J.H. / Shi, Y. / Lovestam, S. / Sew, P.C. / Zhang, X.J. / Kotecha, A. / Bacioglu, M. / Koto, A. / Takao, M. / Spillantini, G.M. / Ghetti, B. / Vidal, R. / Murzin, G.A. / Scheres, H.W.S. / Goedert, M.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025-1013 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_U105184291 United Kingdom
CitationJournal: Acta Neuropathol / Year: 2023
Title: New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.
Authors: Yang Yang / Holly J Garringer / Yang Shi / Sofia Lövestam / Sew Peak-Chew / Xianjun Zhang / Abhay Kotecha / Mehtap Bacioglu / Atsuo Koto / Masaki Takao / Maria Grazia Spillantini / ...Authors: Yang Yang / Holly J Garringer / Yang Shi / Sofia Lövestam / 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 /
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 ...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.
History
DepositionFeb 1, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2May 3, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha-synuclein
C: Alpha-synuclein


Theoretical massNumber of molelcules
Total (without water)28,9522
Polymers28,9522
Non-polymers00
Water0
1
A: Alpha-synuclein
C: Alpha-synuclein

A: Alpha-synuclein
C: Alpha-synuclein

A: Alpha-synuclein
C: Alpha-synuclein


Theoretical massNumber of molelcules
Total (without water)86,8576
Polymers86,8576
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
point symmetry operation2
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(0.999882, -0.015393), (0.015393, 0.999882), (1)1.63605, -1.61105, -4.76672
3generate(0.999882, 0.015393), (-0.015393, 0.999882), (1)-1.61105, 1.63605, 4.76672

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Components

#1: Protein Alpha-synuclein / / Non-A beta component of AD amyloid / Non-A4 component of amyloid precursor / NACP


Mass: 14476.108 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SNCA, NACP, PARK1 / Production host: Escherichia coli (E. coli) / References: UniProt: P37840

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Alpha-synuclein filament assembled in vitro by wild-type and mutant (7 residues insertion) protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0352 / Classification: refinement
EM software
IDNameCategoryDetails
1RELIONparticle selection
2EPUimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9REFMACmodel refinementservalcat
10RELIONinitial Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -179.56 ° / Axial rise/subunit: 2.38 Å / Axial symmetry: C1
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53703 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT
RefinementResolution: 2.7→108.77 Å / Cor.coef. Fo:Fc: 0.756 / SU B: 8.874 / SU ML: 0.178 / ESU R: 0.157
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.44417 --
obs0.44417 49283 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 67.895 Å2
Refinement stepCycle: 1 / Total: 788
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.011790
ELECTRON MICROSCOPYr_bond_other_d0.0010.016808
ELECTRON MICROSCOPYr_angle_refined_deg1.5381.6411066
ELECTRON MICROSCOPYr_angle_other_deg0.6261.5721868
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.6715114
ELECTRON MICROSCOPYr_dihedral_angle_2_deg
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.61510136
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0850.2142
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.02880
ELECTRON MICROSCOPYr_gen_planes_other0.0030.02128
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.0465.987462
ELECTRON MICROSCOPYr_mcbond_other6.0465.986462
ELECTRON MICROSCOPYr_mcangle_it8.9068.99574
ELECTRON MICROSCOPYr_mcangle_other8.9019.007575
ELECTRON MICROSCOPYr_scbond_it9.5877.822328
ELECTRON MICROSCOPYr_scbond_other9.5737.836329
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other15.99610.898493
ELECTRON MICROSCOPYr_long_range_B_refined19.4578.301760
ELECTRON MICROSCOPYr_long_range_B_other19.45278.491760
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.7→2.77 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.042 3613 -
obs--100 %

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