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- EMDB-6183: 3D structure of RepA-WH1 single filaments -

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Basic information

Entry
Database: EMDB / ID: EMD-6183
Title3D structure of RepA-WH1 single filaments
Map dataNegative-staining EM reconstruction of single repA-WH1 filament
Sample
  • Sample: Single RepA-WH1 filament
  • Protein or peptide: RepA
KeywordsRepA-WH1 prionoid / amyloid protofilaments / amyloid assembly
Biological speciesPseudomonas aeruginosa (bacteria)
Methodhelical reconstruction / negative staining / Resolution: 29.0 Å
AuthorsTorreira E / Moreno M / Fuentes-Perez ME / Fernandez C / Martin-Benito J / Moreno-Herrero F / Giraldo R / Llorca O
CitationJournal: Structure / Year: 2015
Title: Amyloidogenesis of bacterial prionoid RepA-WH1 recapitulates dimer to monomer transitions of RepA in DNA replication initiation.
Authors: Eva Torreira / María Moreno-Del Álamo / Maria Eugenia Fuentes-Perez / Cristina Fernández / Jaime Martín-Benito / Fernando Moreno-Herrero / Rafael Giraldo / Oscar Llorca /
Abstract: Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross β sheets. However, structures in which a whole protein domain acts as building block of an ...Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross β sheets. However, structures in which a whole protein domain acts as building block of an amyloid fiber are scarce, in spite of their relevance to understand amyloidogenesis. Here, we use electron microscopy (EM) and atomic force microscopy (AFM) to analyze the structure of amyloid filaments assembled by RepA-WH1, a winged-helix domain from a DNA replication initiator in bacterial plasmids. RepA-WH1 functions as a cytotoxic bacterial prionoid that recapitulates features of mammalian amyloid proteinopathies. RepA are dimers that monomerize at the origin to initiate replication, and we find that RepA-WH1 reproduces this transition to form amyloids. RepA-WH1 assembles double helical filaments by lateral association of a single-stranded precursor built by monomers. Double filaments then associate in mature fibers. The intracellular and cytotoxic RepA-WH1 aggregates might reproduce the hierarchical assembly of human amyloidogenic proteins.
History
DepositionNov 11, 2014-
Header (metadata) releaseNov 26, 2014-
Map releaseNov 26, 2014-
UpdateJan 14, 2015-
Current statusJan 14, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6183.jpg

Downloads & links

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Map

FileDownload / File: emd_6183.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative-staining EM reconstruction of single repA-WH1 filament
Voxel sizeX=Y=Z: 3.75 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.00119907 - 0.10290696
Average (Standard dev.)0.00245138 (±0.01110136)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-48-48-48
Dimensions969696
Spacing969696
CellA=B=C: 360.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.753.753.75
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS-48-48-48
NC/NR/NS969696
D min/max/mean-0.0010.1030.002

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Supplemental data

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Sample components

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Entire : Single RepA-WH1 filament

EntireName: Single RepA-WH1 filament
Components
  • Sample: Single RepA-WH1 filament
  • Protein or peptide: RepA

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Supramolecule #1000: Single RepA-WH1 filament

SupramoleculeName: Single RepA-WH1 filament / type: sample / ID: 1000 / Oligomeric state: single-chain helical filament / Number unique components: 1

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Macromolecule #1: RepA

MacromoleculeName: RepA / type: protein_or_peptide / ID: 1 / Oligomeric state: single-chain helical filament / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
Details: 0.1 M Na2SO4, 40 mM HEPES, 5 mM MgSO4, 7% PEG4000, 3% MPD
StainingType: NEGATIVE
Details: Specimens were adsorbed on glow-discharged copper grids and stained using 2% uranyl acetate for 2 minutes.
GridDetails: 400-mesh copper grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1230
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 41586 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 41586
Sample stageSpecimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using a TVIPS F416 CMOS and the EM-MENU software (TVIPS).
DateSep 1, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Average electron dose: 18 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Each micrograph using BSOFT
Final reconstructionApplied symmetry - Helical parameters - Δz: 12.5 Å
Applied symmetry - Helical parameters - Δ&Phi: 81 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 29.0 Å / Resolution method: OTHER
DetailsThe particles were 2D-classified using CL2D implemented in Xmipp, and further processed using IHRSR. Note: Due to map resolution, the hand is arbitrary.

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Atomic model buiding 1

Initial modelPDB ID:

1hkq

SoftwareName: Chimera
DetailsFitting was carried out both in individual segments and in the whole filament. In this case, helical symmetry was further applied to fill the EM map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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