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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 / : / neutral lipid metabolic process / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / mitochondrial membrane organization ...negative regulation of mitochondrial electron transport, NADH to ubiquinone / : / neutral lipid metabolic process / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / mitochondrial membrane organization / regulation of synaptic vesicle recycling / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / negative regulation of platelet-derived growth factor receptor signaling pathway / positive regulation of protein localization to cell periphery / negative regulation of exocytosis / regulation of glutamate secretion / response to iron(II) ion / SNARE complex assembly / positive regulation of neurotransmitter secretion / dopamine biosynthetic process / regulation of norepinephrine uptake / transporter regulator activity / regulation of locomotion / mitochondrial ATP synthesis coupled electron transport / regulation of macrophage activation / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle priming / negative regulation of microtubule polymerization / synaptic vesicle transport / positive regulation of receptor recycling / dopamine uptake involved in synaptic transmission / protein kinase inhibitor activity / dynein complex binding / regulation of dopamine secretion / negative regulation of thrombin-activated receptor signaling pathway / cuprous ion binding / positive regulation of exocytosis / response to magnesium ion / synaptic vesicle exocytosis / enzyme inhibitor activity / positive regulation of endocytosis / kinesin binding / synaptic vesicle endocytosis / cysteine-type endopeptidase inhibitor activity / negative regulation of serotonin uptake / regulation of presynapse assembly / response to type II interferon / alpha-tubulin binding / supramolecular fiber organization / inclusion body / phospholipid metabolic process / cellular response to copper ion / axon terminus / cellular response to epinephrine stimulus / Hsp70 protein binding / response to interleukin-1 / regulation of microtubule cytoskeleton organization / SNARE binding / positive regulation of release of sequestered calcium ion into cytosol / adult locomotory behavior / negative regulation of protein kinase activity / excitatory postsynaptic potential / fatty acid metabolic process / phosphoprotein binding / protein tetramerization / microglial cell activation / regulation of long-term neuronal synaptic plasticity / synapse organization / ferrous iron binding / protein destabilization / PKR-mediated signaling / phospholipid binding / receptor internalization / tau protein binding / long-term synaptic potentiation / positive regulation of inflammatory response / synaptic vesicle membrane / actin cytoskeleton / actin binding / growth cone / cell cortex / cellular response to oxidative stress / neuron apoptotic process / chemical synaptic transmission / molecular adaptor activity / negative regulation of neuron apoptotic process / response to lipopolysaccharide / histone binding / amyloid fibril formation / lysosome / oxidoreductase activity / transcription cis-regulatory region binding / postsynapse / positive regulation of apoptotic process / Amyloid fiber formation / copper ion binding / response to xenobiotic stimulus / axon / neuronal cell body
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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