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- PDB-7uak: Structure of recombinantly assembled A53E alpha-synuclein fibrils -

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Basic information

Entry
Database: PDB / ID: 7uak
TitleStructure of recombinantly assembled A53E alpha-synuclein fibrils
ComponentsAlpha-synuclein
KeywordsPROTEIN FIBRIL / alpha-synuclein fibrils / A53E mutant / cryo-EM structure / structural protein
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 / regulation of norepinephrine uptake / response to iron(II) ion / dopamine biosynthetic process / SNARE complex assembly / positive regulation of neurotransmitter secretion / regulation of locomotion / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle priming / regulation of macrophage activation / 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 / kinesin binding / positive regulation of endocytosis / mitochondrial ATP synthesis coupled electron transport / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / response to magnesium ion / regulation of presynapse assembly / synaptic vesicle endocytosis / negative regulation of serotonin uptake / alpha-tubulin binding / localization / phospholipid metabolic process / supramolecular fiber organization / axon terminus / 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 / phosphoprotein binding / protein tetramerization / synapse organization / microglial cell activation / regulation of long-term neuronal synaptic plasticity / negative regulation of protein kinase activity / protein destabilization / ferrous iron binding / 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 / phospholipid binding / 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 / actin binding / cell cortex / cellular response to oxidative stress / histone binding / growth cone / postsynapse / chemical synaptic transmission / neuron apoptotic process / negative regulation of neuron apoptotic process / amyloid fibril formation / response to lipopolysaccharide / molecular adaptor activity / oxidoreductase activity / lysosome / 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: 3.38 Å
AuthorsZhou, K. / Zhou, H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)AG 060149 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)1S10OD018111 United States
CitationJournal: J Biol Chem / Year: 2023
Title: Cryo-EM structure of amyloid fibril formed by α-synuclein hereditary A53E mutation reveals a distinct protofilament interface.
Authors: Chuanqi Sun / Kang Zhou / Peter DePaola / Woo Shik Shin / Trae Hillyer / Michael R Sawaya / Ruowei Zhu / Chao Peng / Z Hong Zhou / Lin Jiang /
Abstract: Synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA), have the same pathologic feature of misfolded α-synuclein protein (α-syn) ...Synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA), have the same pathologic feature of misfolded α-synuclein protein (α-syn) accumulation in the brain. PD patients who carry α-syn hereditary mutations tend to have earlier onset and more severe clinical symptoms than sporadic PD patients. Therefore, revealing the effect of hereditary mutations to the α-syn fibril structure can help us understand these synucleinopathies' structural basis. Here, we present a 3.38 Å cryo-electron microscopy structure of α-synuclein fibrils containing the hereditary A53E mutation. The A53E fibril is symmetrically composed of two protofilaments, similar to other fibril structures of WT and mutant α-synuclein. The new structure is distinct from all other synuclein fibrils, not only at the interface between proto-filaments, but also between residues packed within the same proto-filament. A53E has the smallest interface with the least buried surface area among all α-syn fibrils, consisting of only two contacting residues. Within the same protofilament, A53E reveals distinct residue re-arrangement and structural variation at a cavity near its fibril core. Moreover, the A53E fibrils exhibit slower fibril formation and lower stability compared to WT and other mutants like A53T and H50Q, while also demonstrate strong cellular seeding in α-synuclein biosensor cells and primary neurons. In summary, our study aims to highlight structural differences - both within and between the protofilaments of A53E fibrils - and interpret fibril formation and cellular seeding of α-synuclein pathology in disease, which could further our understanding of the structure-activity relationship of α-synuclein mutants.
History
DepositionMar 13, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author / Item: _citation.journal_volume / _citation_author.name
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Alpha-synuclein
B: Alpha-synuclein
C: Alpha-synuclein
D: Alpha-synuclein
E: Alpha-synuclein
F: Alpha-synuclein


Theoretical massNumber of molelcules
Total (without water)87,2056
Polymers87,2056
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 14534.146 Da / Num. of mol.: 6 / Mutation: A53E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SNCA, NACP, PARK1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 A53E mutant / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 59 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.4
Buffer componentConc.: 15 mM / Name: tetrabutylphosphonium bromide / Formula: TBPB
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 48 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2767
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 5 / Used frames/image: 2-34

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.6particle selection
2SerialEMimage acquisition
4CTFFIND4.1.5CTF correction
7Coot0.8.9model fitting
12RELION3.1.23D reconstruction
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 179.504 ° / Axial rise/subunit: 2.418 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 46626
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31916 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 125 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6LRQ

6lrq


Pdb chain-ID: A / Accession code: 6LRQ / Source name: PDB / Type: experimental model

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