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- EMDB-4392: In situ subtomogram average of a bacterial encapsulin expressed i... -

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Basic information

Entry
Database: EMDB / ID: EMD-4392
TitleIn situ subtomogram average of a bacterial encapsulin expressed in HEK293T cells (no iron treatment)
Map datastructure of EncABCD without iron treatment
Sample
  • Complex: Encapsulin from M. xanthus in its T = 3 configuration
Biological speciesMyxococcus xanthus (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 19.6 Å
AuthorsErdmann PS / Plitzko JM
CitationJournal: Nat Commun / Year: 2018
Title: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering.
Authors: Felix Sigmund / Christoph Massner / Philipp Erdmann / Anja Stelzl / Hannes Rolbieski / Mitul Desai / Sarah Bricault / Tobias P Wörner / Joost Snijder / Arie Geerlof / Helmut Fuchs / Martin ...Authors: Felix Sigmund / Christoph Massner / Philipp Erdmann / Anja Stelzl / Hannes Rolbieski / Mitul Desai / Sarah Bricault / Tobias P Wörner / Joost Snijder / Arie Geerlof / Helmut Fuchs / Martin Hrabĕ de Angelis / Albert J R Heck / Alan Jasanoff / Vasilis Ntziachristos / Jürgen Plitzko / Gil G Westmeyer /
Abstract: We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size- ...We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.
History
DepositionApr 25, 2018-
Header (metadata) releaseMay 30, 2018-
Map releaseMay 30, 2018-
UpdateMay 30, 2018-
Current statusMay 30, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4392.png

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Map

FileDownload / File: emd_4392.map.gz / Format: CCP4 / Size: 23.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of EncABCD without iron treatment
Voxel sizeX=Y=Z: 3.42 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.17286582 - 0.5678482
Average (Standard dev.)0.0042168545 (±0.09847571)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions184184184
Spacing184184184
CellA=B=C: 629.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.423.423.42
M x/y/z184184184
origin x/y/z0.0000.0000.000
length x/y/z629.280629.280629.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS184184184
D min/max/mean-0.1730.5680.004

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Supplemental data

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Sample components

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Entire : Encapsulin from M. xanthus in its T = 3 configuration

EntireName: Encapsulin from M. xanthus in its T = 3 configuration
Components
  • Complex: Encapsulin from M. xanthus in its T = 3 configuration

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Supramolecule #1: Encapsulin from M. xanthus in its T = 3 configuration

SupramoleculeName: Encapsulin from M. xanthus in its T = 3 configuration / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Myxococcus xanthus (bacteria)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T
Molecular weightTheoretical: 1.2 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7 / Details: cells were plunge frozen in DMEM with 10% FBS
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV / Details: blot force: 10 blot time: 10.
Detailsthe sample was prepared by focused ion beam milling of vitrified HEK293T cells

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: -5.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 6 / Number images used: 1600 / Reference model: reference free / Method: volumes picked interactively / Software - Name: PyTom (ver. 0.97)
Details: a refrence was constructed from ~ 100 manually picked and aligned particles
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.5), RELION (ver. 2.1))
Final 3D classificationSoftware - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 19.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number subtomograms used: 317

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