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- EMDB-4879: QtEnc_E.coli -

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Basic information

Entry
Database: EMDB / ID: EMD-4879
TitleQtEnc_E.coli
Map dataMap of QtEnc expressen in E.coli
Sample
  • Organelle or cellular component: QtEnc
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKube M / Westmeyer G
CitationJournal: ACS Nano / Year: 2019
Title: Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters.
Authors: Felix Sigmund / Susanne Pettinger / Massimo Kube / Fabian Schneider / Martina Schifferer / Steffen Schneider / Maria V Efremova / Jesús Pujol-Martí / Michaela Aichler / Axel Walch / Thomas ...Authors: Felix Sigmund / Susanne Pettinger / Massimo Kube / Fabian Schneider / Martina Schifferer / Steffen Schneider / Maria V Efremova / Jesús Pujol-Martí / Michaela Aichler / Axel Walch / Thomas Misgeld / Hendrik Dietz / Gil G Westmeyer /
Abstract: Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance ...Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of (Qt), which in combination with the recently characterized encapsulin system from (Mx) enables multiplexed gene reporter imaging conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM a deep learning classifier to enable automated multiplexed EM gene reporter imaging.
History
DepositionApr 17, 2019-
Header (metadata) releaseJun 19, 2019-
Map releaseJun 26, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0621
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0621
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4879.png

Downloads & links

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Map

FileDownload / File: emd_4879.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of QtEnc expressen in E.coli
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.0621 / Movie #1: 0.0621
Minimum - Maximum-0.07489338 - 0.19488823
Average (Standard dev.)0.00004373512 (-)
SymmetrySpace group: 0
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 552.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z243.000243.000243.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS-90-90-90
NC/NR/NS180180180
D min/max/mean-0.2760.3510.001

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Supplemental data

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Sample components

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Entire : QtEnc

EntireName: QtEnc
Components
  • Organelle or cellular component: QtEnc

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Supramolecule #1: QtEnc

SupramoleculeName: QtEnc / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 56144
FSC plot (resolution estimation)

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