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- PDB-7ovt: major seeded in vitro fibril morphology from murine SAA1.1 protein -

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Basic information

Entry
Database: PDB / ID: 7ovt
Titlemajor seeded in vitro fibril morphology from murine SAA1.1 protein
ComponentsSerum amyloid A-2 protein
KeywordsPROTEIN FIBRIL / systemic amyloidosis / seeded / misfolding disease / inflammation / prion
Function / homologySerum amyloid A protein / : / Serum amyloid A protein / Serum amyloid A proteins signature. / Serum amyloid A proteins / response to stilbenoid / high-density lipoprotein particle / acute-phase response / Serum amyloid A-2 protein
Function and homology information
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsHeerde, T. / Schmidt, M. / Faendrich, M.
Funding support Germany, 6items
OrganizationGrant numberCountry
German Research Foundation (DFG)DFG SCHM 3276/1 Germany
German Research Foundation (DFG)DFG FA 456/23 Germany
German Research Foundation (DFG)DFG FA 456/27 Germany
German Research Foundation (DFG)DFG HA 7138/3 Germany
German Research Foundation (DFG)DFG HA 7138/2 Germany
German Research Foundation (DFG)CRC 1279 project A03 Germany
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding.
Authors: Thomas Heerde / Matthies Rennegarbe / Alexander Biedermann / Dilan Savran / Peter B Pfeiffer / Manuel Hitzenberger / Julian Baur / Ioana Puscalau-Girtu / Martin Zacharias / Nadine Schwierz / ...Authors: Thomas Heerde / Matthies Rennegarbe / Alexander Biedermann / Dilan Savran / Peter B Pfeiffer / Manuel Hitzenberger / Julian Baur / Ioana Puscalau-Girtu / Martin Zacharias / Nadine Schwierz / Christian Haupt / Matthias Schmidt / Marcus Fändrich /
Abstract: Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the ...Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.
History
DepositionJun 15, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Assembly

Deposited unit
A: Serum amyloid A-2 protein
B: Serum amyloid A-2 protein
C: Serum amyloid A-2 protein
D: Serum amyloid A-2 protein
E: Serum amyloid A-2 protein
F: Serum amyloid A-2 protein
G: Serum amyloid A-2 protein
H: Serum amyloid A-2 protein
I: Serum amyloid A-2 protein
J: Serum amyloid A-2 protein
K: Serum amyloid A-2 protein
L: Serum amyloid A-2 protein


Theoretical massNumber of molelcules
Total (without water)139,47212
Polymers139,47212
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area60920 Å2
ΔGint-157 kcal/mol
Surface area26960 Å2

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Components

#1: Protein
Serum amyloid A-2 protein


Mass: 11622.629 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Details: amyloid fibril / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Saa2 / Production host: Escherichia coli (E. coli) / References: UniProt: P05367

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Murine serum amyloid A1 (SAA1) amyloid fibril / Type: COMPLEX
Details: in vitro murine SAA amyloid fibril morphology i; Seeded with ex vivo material
Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5 / Details: 10mM Tris(hydroxymethyl)aminomethane (Tris)
Buffer componentConc.: 10 mM / Name: Tris(hydroxymethyl)aminomethane / Formula: (HOCH2)3CNH2
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 96 %

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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 / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
7Coot0.8.9model fitting
9PHENIX1.16-3549model refinement
12RELION3.0.4classification
13RELION3.0.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 179.425 ° / Axial rise/subunit: 2.4 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 141159
3D reconstructionResolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107856 / Symmetry type: HELICAL
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL / Target criteria: correlation coefficient

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