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- EMDB-13089: major seeded in vitro fibril morphology from murine SAA1.1 protein -

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Basic information

Entry
Database: EMDB / ID: EMD-13089
Titlemajor seeded in vitro fibril morphology from murine SAA1.1 protein
Map dataMasked post-processed density map of the reconstructed murine SAA1.1 fibril
Sample
  • Complex: Murine serum amyloid A1 (SAA1) amyloid fibril
    • Protein or peptide: Serum amyloid A-2 protein
Keywordssystemic amyloidosis / seeded / misfolding disease / inflammation / prion / protein fibril
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)
Methodhelical reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsHeerde T / Schmidt M
Funding support Germany, 6 items
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, 2021-
Header (metadata) releaseFeb 2, 2022-
Map releaseFeb 2, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0314
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0314
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ovt
  • Surface level: 0.0314
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ovt
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13089.png

Downloads & links

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Map

FileDownload / File: emd_13089.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked post-processed density map of the reconstructed murine SAA1.1 fibril
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 300 pix.
= 312. Å		1.04 Å/pix.
x 300 pix.
= 312. Å		1.04 Å/pix.
x 300 pix.
= 312. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0314 / Movie #1: 0.0314
Minimum - Maximum-0.07857801 - 0.15138999
Average (Standard dev.)0.00007501277 (±0.002433123)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 312.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z312.000312.000312.000
α/β/γ90.00090.00090.000
start NX/NY/NZ258279248
NX/NY/NZ8855101
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0790.1510.000

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Supplemental data

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Mask #1

Fileemd_13089_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Second half map of the reconstructed fibril

Fileemd_13089_half_map_1.map
AnnotationSecond half map of the reconstructed fibril
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: First half map of the reconstructed fibril

Fileemd_13089_half_map_2.map
AnnotationFirst half map of the reconstructed fibril
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Murine serum amyloid A1 (SAA1) amyloid fibril

EntireName: Murine serum amyloid A1 (SAA1) amyloid fibril
Components
  • Complex: Murine serum amyloid A1 (SAA1) amyloid fibril
    • Protein or peptide: Serum amyloid A-2 protein

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Supramolecule #1: Murine serum amyloid A1 (SAA1) amyloid fibril

SupramoleculeName: Murine serum amyloid A1 (SAA1) amyloid fibril / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: in vitro murine SAA amyloid fibril morphology i; Seeded with ex vivo material
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Serum amyloid A-2 protein

MacromoleculeName: Serum amyloid A-2 protein / type: protein_or_peptide / ID: 1 / Details: amyloid fibril / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.622629 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GFFSFIGEAF QGAGDMWRAY TDMKEAGWKD GDKYFHARGN YDAAQRGPGG VWAAEKISDA RESFQEFFGR GHEDTMADQE ANRHGRSGK DPNYYRPPGL PAKY

UniProtKB: Serum amyloid A-2 protein

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 8.5 / Component - Concentration: 10.0 mM / Component - Formula: (HOCH2)3CNH2 / Component - Name: Tris(hydroxymethyl)aminomethane / Details: 10mM Tris(hydroxymethyl)aminomethane (Tris)
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 96 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 12.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 2.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 179.425 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.4) / Number images used: 107856
Segment selectionNumber selected: 141159
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6dso

Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Target criteria: correlation coefficient
Output model

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

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