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- PDB-9fyp: Cryo EM structure of the type 3B polymorph of alpha-synuclein at ... -

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Basic information

Entry
Database: PDB / ID: 9fyp
TitleCryo EM structure of the type 3B polymorph of alpha-synuclein at low pH.
ComponentsAlpha-synuclein
KeywordsPROTEIN FIBRIL / amyloid / polymorphism
Function / homology
Function and homology information


regulation of phospholipase activity / : / 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 ...regulation of phospholipase activity / : / 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 / negative regulation of transporter activity / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / regulation of synaptic vesicle recycling / 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 / regulation of locomotion / synaptic vesicle priming / mitochondrial ATP synthesis coupled electron transport / regulation of macrophage activation / positive regulation of inositol phosphate biosynthetic process / negative regulation of microtubule polymerization / synaptic vesicle transport / dynein complex binding / positive regulation of receptor recycling / dopamine uptake involved in synaptic transmission / protein kinase inhibitor activity / regulation of dopamine secretion / negative regulation of thrombin-activated receptor signaling pathway / cuprous ion binding / positive regulation of endocytosis / synaptic vesicle exocytosis / positive regulation of exocytosis / response to magnesium ion / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / kinesin binding / synaptic vesicle endocytosis / regulation of presynapse assembly / response to type II interferon / negative regulation of serotonin uptake / alpha-tubulin binding / inclusion body / supramolecular fiber organization / phospholipid metabolic process / cellular response to copper ion / cellular response to epinephrine stimulus / axon terminus / Hsp70 protein binding / response to interleukin-1 / SNARE binding / positive regulation of release of sequestered calcium ion into cytosol / adult locomotory behavior / excitatory postsynaptic potential / fatty acid metabolic process / positive regulation of protein serine/threonine kinase activity / phosphoprotein binding / negative regulation of protein kinase activity / protein tetramerization / long-term synaptic potentiation / regulation of transmembrane transporter activity / microglial cell activation / regulation of long-term neuronal synaptic plasticity / synapse organization / ferrous iron binding / positive regulation of peptidyl-serine phosphorylation / protein destabilization / PKR-mediated signaling / tau protein binding / receptor internalization / phospholipid binding / synaptic vesicle membrane / positive regulation of inflammatory response / actin cytoskeleton / actin binding / growth cone / cell cortex / cellular response to oxidative stress / chemical synaptic transmission / neuron apoptotic process / molecular adaptor activity / response to lipopolysaccharide / negative regulation of neuron apoptotic process / histone binding / amyloid fibril formation / lysosome / oxidoreductase activity / transcription cis-regulatory region binding / postsynapse / positive regulation of apoptotic process / copper ion binding / response to xenobiotic stimulus
Similarity search - Function
Synuclein / Alpha-synuclein / Synuclein
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.23 Å
AuthorsFrey, L. / Qureshi, B.M. / Kwiatkowski, W. / Rhyner, D. / Greenwald, J. / Riek, R.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
Other government
Citation
Journal: Elife / Year: 2024
Title: On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation.
Authors: Lukas Frey / Dhiman Ghosh / Bilal M Qureshi / David Rhyner / Ricardo Guerrero-Ferreira / Aditya Pokharna / Witek Kwiatkowski / Tetiana Serdiuk / Paola Picotti / Roland Riek / Jason Greenwald /
Abstract: The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical ...The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.
#1: Journal: Elife / Year: 2024
Title: On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation.
Authors: Lukas Frey / Dhiman Ghosh / Bilal M Qureshi / David Rhyner / Ricardo Guerrero-Ferreira / Aditya Pokharna / Witek Kwiatkowski / Tetiana Serdiuk / Paola Picotti / Roland Riek / Jason Greenwald /
Abstract: The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical ...The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.
#2: Journal: Elife / Year: 2024
Title: On the pH-dependence of alpha-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation
Authors: Frey, L. / Ghosh, D. / Qureshi, B.M. / Rhyner, D. / Guerrero-Ferreira, R. / Pokharna, A. / Kwiatkowski, W. / Serdiuk, T. / Picotti, P. / Riek, R. / Greenwald, J.
History
DepositionJul 3, 2024Deposition site: PDBE / Processing site: PDBE
SupersessionJul 17, 2024ID: 8PIC
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update
Revision 1.2Jan 22, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
I: Alpha-synuclein
J: Alpha-synuclein
K: Alpha-synuclein
L: Alpha-synuclein
M: Alpha-synuclein
N: Alpha-synuclein
O: Alpha-synuclein
P: Alpha-synuclein
Q: Alpha-synuclein
R: Alpha-synuclein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,11620
Polymers144,76110
Non-polymers35510
Water2,162120
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 / Non-A beta component of AD amyloid / Non-A4 component of amyloid precursor / NACP


Mass: 14476.108 Da / Num. of mol.: 10
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
#2: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Cl
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 120 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN

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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 amyloid fibril / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 5.8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMPhosphatePO41
2137 mMSodium ChlorideNaCl1
32.7 mMPotassiu ChlorideKCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 800 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: 1 sec. / Electron dose: 62 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 7729
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
12RELION4.0.0classification
13RELION4.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 179.5 ° / Axial rise/subunit: 2.37 Å / Axial symmetry: C1
3D reconstructionResolution: 2.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 178710 / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 36.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00494440
ELECTRON MICROSCOPYf_angle_d0.6366000
ELECTRON MICROSCOPYf_chiral_restr0.0606780
ELECTRON MICROSCOPYf_plane_restr0.0027750
ELECTRON MICROSCOPYf_dihedral_angle_d11.63731510

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