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- PDB-8qpz: CryoEM structure of recombinant DeltaN7 alpha-synuclein in PBS -

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Basic information

Entry
Database: PDB / ID: 8qpz
TitleCryoEM structure of recombinant DeltaN7 alpha-synuclein in PBS
ComponentsAlpha-synuclein
KeywordsPROTEIN FIBRIL / synuclein / Parkinson's disease / neurodegeneration / amyloid / helical / fibril
Function / homology
Function and homology information


negative regulation of mitochondrial electron transport, NADH to ubiquinone / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / response to desipramine / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / regulation of synaptic vesicle recycling / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process ...negative regulation of mitochondrial electron transport, NADH to ubiquinone / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / response to desipramine / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / regulation of synaptic vesicle recycling / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / positive regulation of protein localization to cell periphery / negative regulation of exocytosis / dopamine biosynthetic process / dopamine uptake involved in synaptic transmission / response to iron(II) ion / negative regulation of dopamine metabolic process / negative regulation of platelet-derived growth factor receptor signaling pathway / SNARE complex assembly / negative regulation of thrombin-activated receptor signaling pathway / Lewy body / negative regulation of microtubule polymerization / synaptic vesicle priming / regulation of norepinephrine uptake / transporter regulator activity / protein kinase inhibitor activity / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle transport / regulation of dopamine secretion / positive regulation of receptor recycling / cuprous ion binding / positive regulation of exocytosis / nuclear outer membrane / dynein complex binding / synaptic transmission, dopaminergic / synaptic vesicle exocytosis / response to magnesium ion / positive regulation of endocytosis / negative regulation of serotonin uptake / kinesin binding / cysteine-type endopeptidase inhibitor activity / regulation of presynapse assembly / synaptic vesicle endocytosis / alpha-tubulin binding / beta-tubulin binding / phospholipase binding / behavioral response to cocaine / supramolecular fiber organization / cellular response to fibroblast growth factor stimulus / response to type II interferon / cellular response to epinephrine stimulus / inclusion body / Hsp70 protein binding / response to interleukin-1 / axon terminus / cellular response to copper ion / positive regulation of release of sequestered calcium ion into cytosol / enzyme inhibitor activity / regulation of microtubule cytoskeleton organization / SNARE binding / glutathione metabolic process / protein tetramerization / protein sequestering activity / phosphoprotein binding / receptor internalization / tubulin binding / microglial cell activation / ferrous iron binding / phospholipid binding / PKR-mediated signaling / synapse organization / protein destabilization / tau protein binding / enzyme activator activity / terminal bouton / positive regulation of inflammatory response / actin cytoskeleton / synaptic vesicle membrane / growth cone / actin binding / cellular response to oxidative stress / response to lipopolysaccharide / histone binding / cell cortex / microtubule binding / amyloid fibril formation / negative regulation of neuron apoptotic process / mitochondrial outer membrane / lysosome / oxidoreductase activity / mitochondrial inner membrane / transcription cis-regulatory region binding / positive regulation of apoptotic process / ribosome / mitochondrial matrix / Amyloid fiber formation / copper ion binding / protein domain specific binding / axon / neuronal cell body / calcium ion binding
Similarity search - Function
Synuclein / Alpha-synuclein / Synuclein
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsThacker, D. / Wilkinson, M. / Dewison, K.M. / Ranson, N.A. / Brockwell, D.J. / Radford, S.E.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust094232/Z/10/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N013840/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Residues 2 to 7 of α-synuclein regulate amyloid formation via lipid-dependent and lipid-independent pathways.
Authors: Katherine M Dewison / Benjamin Rowlinson / Jonathan M Machin / Joel A Crossley / Dev Thacker / Martin Wilkinson / Sabine M Ulamec / G Nasir Khan / Neil A Ranson / Patricija van Oosten-Hawle ...Authors: Katherine M Dewison / Benjamin Rowlinson / Jonathan M Machin / Joel A Crossley / Dev Thacker / Martin Wilkinson / Sabine M Ulamec / G Nasir Khan / Neil A Ranson / Patricija van Oosten-Hawle / David J Brockwell / Sheena E Radford /
Abstract: Amyloid formation by α-synuclein (αSyn) occurs in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation ...Amyloid formation by α-synuclein (αSyn) occurs in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate αSyn amyloid fibril formation will not only provide mechanistic insight but may also reveal targets to prevent and treat disease. Previous investigations have identified several regions of αSyn to be important in the regulation of amyloid formation, including the non-amyloid-β component (NAC), P1 region (residues 36 to 42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of αSyn for both its physiological and pathological roles. Here, the role of residues 2 to 7 in the N-terminal region of αSyn is investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Together, the results demonstrate the involvement of residues 2 to 7 of αSyn in amyloid formation, revealing a target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed.
History
DepositionOct 3, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release

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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
G: Alpha-synuclein
H: Alpha-synuclein
I: Alpha-synuclein
J: Alpha-synuclein
K: Alpha-synuclein
L: Alpha-synuclein


Theoretical massNumber of molelcules
Total (without water)165,56712
Polymers165,56712
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Alpha-synuclein


Mass: 13797.286 Da / Num. of mol.: 12 / Mutation: deltaN7 (methionine kept for expression)
Source method: isolated from a genetically manipulated source
Details: DeltaN7, technically residues 2-7 are deleted as the N-terminal Methionine was required for bacterial expression.
Source: (gene. exp.) Homo sapiens (human) / Gene: SNCA / Variant: DeltaN7 / Plasmid: pET23a / Production host: Escherichia coli BL21(DE3) (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 DeltaN7 amyloid fibrils / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Details: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4; pH 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 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: 2600 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5464
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPU3image acquisition
4CTFFIND4.16CTF correction
7Coot0.9.8model fitting
9RELION4initial Euler assignment
10RELION4final Euler assignment
11RELION4classification
12RELION43D reconstruction
13PHENIX1.17.1_3660:model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 179.43 ° / Axial rise/subunit: 2.44 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 315777
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12348 / Symmetry type: HELICAL
Atomic model buildingB value: 106 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: see methods, rigid body docked chain of PDB: 6osl, manual fitting in coot, real-space refine in phenix
Atomic model buildingPDB-ID: 6osl

6osl


Pdb chain-ID: A / Accession code: 6osl / Chain residue range: 42-92 / Pdb chain residue range: 42-92 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044116
ELECTRON MICROSCOPYf_angle_d0.6945580
ELECTRON MICROSCOPYf_dihedral_angle_d19.0921380
ELECTRON MICROSCOPYf_chiral_restr0.049768
ELECTRON MICROSCOPYf_plane_restr0.002696

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