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- EMDB-18570: CryoEM structure of recombinant DeltaN7 alpha-synuclein in PBS -

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Basic information

Entry
Database: EMDB / ID: EMD-18570
TitleCryoEM structure of recombinant DeltaN7 alpha-synuclein in PBS
Map dataPostprocessed sharpened cryoEM map for the aSyn-DN7 fibril structure
Sample
  • Complex: alpha-synuclein DeltaN7 amyloid fibrils
    • Protein or peptide: Alpha-synuclein
Keywordssynuclein / Parkinson's disease / neurodegeneration / amyloid / helical / fibril / PROTEIN 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 / negative regulation of chaperone-mediated autophagy / regulation of synaptic vesicle recycling / 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 / negative regulation of chaperone-mediated autophagy / regulation of synaptic vesicle recycling / 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 / negative regulation of dopamine metabolic process / response to iron(II) ion / negative regulation of platelet-derived growth factor receptor signaling pathway / SNARE complex assembly / negative regulation of thrombin-activated receptor signaling pathway / negative regulation of microtubule polymerization / synaptic vesicle priming / synaptic vesicle transport / Lewy body / synaptic vesicle exocytosis / regulation of norepinephrine uptake / transporter regulator activity / positive regulation of inositol phosphate biosynthetic process / protein kinase inhibitor activity / 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 / response to magnesium ion / positive regulation of endocytosis / negative regulation of serotonin uptake / cysteine-type endopeptidase inhibitor activity / kinesin binding / regulation of presynapse assembly / alpha-tubulin binding / synaptic vesicle endocytosis / beta-tubulin binding / behavioral response to cocaine / phospholipase binding / cellular response to fibroblast growth factor stimulus / supramolecular fiber organization / response to type II interferon / cellular response to epinephrine stimulus / inclusion body / response to interleukin-1 / Hsp70 protein binding / positive regulation of release of sequestered calcium ion into cytosol / cellular response to copper ion / axon terminus / enzyme inhibitor activity / glutathione metabolic process / SNARE binding / protein tetramerization / regulation of microtubule cytoskeleton organization / receptor internalization / phosphoprotein binding / microglial cell activation / protein destabilization / protein sequestering activity / tubulin binding / ferrous iron binding / synapse organization / phospholipid binding / PKR-mediated signaling / tau protein binding / enzyme activator activity / positive regulation of inflammatory response / terminal bouton / actin cytoskeleton / synaptic vesicle membrane / negative regulation of neuron apoptotic process / response to lipopolysaccharide / growth cone / actin binding / cellular response to oxidative stress / histone binding / cell cortex / microtubule binding / amyloid fibril formation / 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)
Methodhelical reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsThacker D / Wilkinson M / Dewison KM / Ranson NA / Brockwell DJ / Radford SE
Funding support United Kingdom, 2 items
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, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18570.png

Downloads & links

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Map

FileDownload / File: emd_18570.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed sharpened cryoEM map for the aSyn-DN7 fibril structure
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å		0.95 Å/pix.
x 300 pix.
= 285. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.95 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.026463725 - 0.06276156
Average (Standard dev.)0.00036333947 (±0.002465426)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 285.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: halfmap2 for the aSyn-DN7 fibril structure

Fileemd_18570_half_map_1.map
Annotationhalfmap2 for the aSyn-DN7 fibril structure
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1 for the aSyn-DN7 fibril structure

Fileemd_18570_half_map_2.map
Annotationhalfmap1 for the aSyn-DN7 fibril structure
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : alpha-synuclein DeltaN7 amyloid fibrils

EntireName: alpha-synuclein DeltaN7 amyloid fibrils
Components
  • Complex: alpha-synuclein DeltaN7 amyloid fibrils
    • Protein or peptide: Alpha-synuclein

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Supramolecule #1: alpha-synuclein DeltaN7 amyloid fibrils

SupramoleculeName: alpha-synuclein DeltaN7 amyloid fibrils / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Alpha-synuclein

MacromoleculeName: Alpha-synuclein / type: protein_or_peptide / ID: 1
Details: DeltaN7, technically residues 2-7 are deleted as the N-terminal Methionine was required for bacterial expression.
Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.797286 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MLSKAKEGVV AAAEKTKQGV AEAAGKTKEG VLYVGSKTKE GVVHGVATVA EKTKEQVTNV GGAVVTGVTA VAQKTVEGAG SIAAATGFV KKDQLGKNEE GAPQEGILED MPVDPDNEAY EMPSEEGYQD YEPEA

UniProtKB: Alpha-synuclein

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
Details: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4; pH 7.4
GridMaterial: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 5464 / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 2.44 Å
Applied symmetry - Helical parameters - Δ&Phi: 179.43 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 12348
Segment selectionNumber selected: 315777 / Software - Name: Topaz
Startup modelType of model: INSILICO MODEL
Details: relion_helix_inimodel2d from a single 2D class average
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6osl


Chain - Chain ID: A / Chain - Residue range: 42-92 / Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailssee methods, rigid body docked chain of PDB: 6osl, manual fitting in coot, real-space refine in phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 106 / Target criteria: cross-correlation coefficient
Output model

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

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