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- EMDB-11598: Shotgun EM of Mycobacterial protein complexes during stationary p... -

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Basic information

Entry
Database: EMDB / ID: EMD-11598
TitleShotgun EM of Mycobacterial protein complexes during stationary phase stress.
Map dataLow-pass filtered
Sample
  • Complex: Encapsulin containing DyP peroxidase cargo protein.
    • Protein or peptide: Encapsulin
    • Protein or peptide: DyP-peroxidase
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 27.0 Å
AuthorsWoodward JD / Kirykowicz AM
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Science and Technology Funding CouncilST/R002754/1 United Kingdom
CitationJournal: Curr Res Struct Biol / Year: 2020
Title: Shotgun EM of mycobacterial protein complexes during stationary phase stress.
Authors: Angela M Kirykowicz / Jeremy D Woodward /
Abstract: There is little structural information about the protein complexes conferring resistance in to anti-microbial oxygen and nitrogen radicals in the phagolysosome. Here, we expose the model ...There is little structural information about the protein complexes conferring resistance in to anti-microbial oxygen and nitrogen radicals in the phagolysosome. Here, we expose the model Mycobacterium, to simulated oxidative-stress conditions and apply a shotgun EM method for the structural detection of the resulting protein assemblies. We identified: glutamine synthetase I, essential for virulence; bacterioferritin A, critical for iron regulation; aspartyl aminopeptidase M18, a protease; and encapsulin, which produces a cage-like structure to enclose cargo proteins. After further investigation, we found that encapsulin carries dye-decolourising peroxidase, a protein antioxidant, as its primary cargo under the conditions tested.
History
DepositionAug 6, 2020-
Header (metadata) releaseSep 9, 2020-
Map releaseSep 9, 2020-
UpdateJul 21, 2021-
Current statusJul 21, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0983
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0983
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11598.png

Downloads & links

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Map

FileDownload / File: emd_11598.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLow-pass filtered
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.84 Å/pix.
x 128 pix.
= 491.52 Å		3.84 Å/pix.
x 128 pix.
= 491.52 Å		3.84 Å/pix.
x 128 pix.
= 491.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0983 / Movie #1: 0.0983
Minimum - Maximum-0.19168611 - 0.28222138
Average (Standard dev.)0.0064011784 (±0.0314526)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 491.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.843.843.84
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z491.520491.520491.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1920.2820.006

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Supplemental data

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Sample components

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Entire : Encapsulin containing DyP peroxidase cargo protein.

EntireName: Encapsulin containing DyP peroxidase cargo protein.
Components
  • Complex: Encapsulin containing DyP peroxidase cargo protein.
    • Protein or peptide: Encapsulin
    • Protein or peptide: DyP-peroxidase

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Supramolecule #1: Encapsulin containing DyP peroxidase cargo protein.

SupramoleculeName: Encapsulin containing DyP peroxidase cargo protein. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: produced by Mycobacterium smegmatis under stationary phase stress
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 1.9 MDa

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Macromolecule #1: Encapsulin

MacromoleculeName: Encapsulin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MNNLYRDLAP ITESAWAEIE LEATRTFKRH IAGRRVVDVS GPNGPTTASV STGHLLDVSP PGDGVIAHLR DAKPLVRLRV PFTVARRDID DVERGSQDSD WDPVKDAAKK LAFVEDRAIF EGYAAASIEG IRSSSSNPAL ALPDDAREIP DVIAQALSEL RLAGVDGPYS ...String:
MNNLYRDLAP ITESAWAEIE LEATRTFKRH IAGRRVVDVS GPNGPTTASV STGHLLDVSP PGDGVIAHLR DAKPLVRLRV PFTVARRDID DVERGSQDSD WDPVKDAAKK LAFVEDRAIF EGYAAASIEG IRSSSSNPAL ALPDDAREIP DVIAQALSEL RLAGVDGPYS VLLSAETYTK VSETTAHGYP IREHINRLVD GEIIWAPAID GAFVLSTRGG DFDLQLGTDV SIGYLSHDAE VVHLYMEETM TFLCYTAEAS VALTP

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Macromolecule #2: DyP-peroxidase

MacromoleculeName: DyP-peroxidase / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: dye decolorizing peroxidase
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: MPAPQPQPVL APLTPAAVFL VATIDEGQEA TVYDALPDIS GLVRAIGFRD PAKRLSAITS IGSDAWDRLF SGPRPAELHP FREIDGGRHH APATPGDLLF HLRAESMDVC FELATKLVEA MSGAITIVDE THGFRFFDNR DLMGFVDGTE NPDGNLAVVA TQIGDEDPDF ...String:
MPAPQPQPVL APLTPAAVFL VATIDEGQEA TVYDALPDIS GLVRAIGFRD PAKRLSAITS IGSDAWDRLF SGPRPAELHP FREIDGGRHH APATPGDLLF HLRAESMDVC FELATKLVEA MSGAITIVDE THGFRFFDNR DLMGFVDGTE NPDGNLAVVA TQIGDEDPDF AGGCYVHVQK YLHDMASWNS LSVEEQERVI GRTKLDDIEL DDDVKPANSH VALNVIEDED GNELKIIRHN MPFGEIGKGE FGTYYIGYSR TPSVTERMLD NMFIGDPPGN TDRILDFSTA ITGGLFFTPT VDFLDDPPPL PSEDDRAEPA SAPSADPVHT DGSLGIGSLK GTR

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3TRIS-HCl
300.0 mMNaClSodium Chloride
StainingType: NEGATIVE / Material: Uranyl acetate
GridModel: Homemade / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.1 kPa
DetailsPartially fractionated cell lysate

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 181 / Average exposure time: 5.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 1.0 µm / Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe images were assessed for drift and astigmatism. No CTF correction was applied.
Particle selectionNumber selected: 3306
Details: Laplacian of Gaussian Autopicking was used with a wide diameter range 10 nm - 30 nm to select a large number of different particles. These were 2D classified and sorted.
Startup modelType of model: OTHER
Details: Initial model was generated by stochastic gradient descent.
Final reconstructionNumber classes used: 10 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 1828
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 50 / Avg.num./class: 66 / Software - Name: RELION (ver. 3.1)
Details: Classes belonging to this particle were sorted using SLICEM and selected.
FSC plot (resolution estimation)

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