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- EMDB-11597: Shotgun EM of Mycobacterial protein complexes during stationary p... -

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Basic information

Entry
Database: EMDB / ID: EMD-11597
TitleShotgun EM of Mycobacterial protein complexes during stationary phase stress.
Map dataLow-pass filtered according to FSC
Sample
  • Complex: Encapsulin
    • Protein or peptide: Encapsulin
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 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) releaseAug 19, 2020-
Map releaseAug 19, 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: 0.0565
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0565
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11597.png

Downloads & links

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Map

FileDownload / File: emd_11597.map.gz / Format: CCP4 / Size: 2.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLow-pass filtered according to FSC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.84 Å/pix.
x 84 pix.
= 322.56 Å		3.84 Å/pix.
x 84 pix.
= 322.56 Å		3.84 Å/pix.
x 84 pix.
= 322.56 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: 0.0565 / Movie #1: 0.0565
Minimum - Maximum-0.052444275 - 0.09788644
Average (Standard dev.)0.0074810116 (±0.022498777)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions848484
Spacing848484
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.843.843.84
M x/y/z848484
origin x/y/z0.0000.0000.000
length x/y/z322.560322.560322.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS848484
D min/max/mean-0.0520.0980.007

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Supplemental data

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Sample components

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Entire : Encapsulin

EntireName: Encapsulin
Components
  • Complex: Encapsulin
    • Protein or peptide: Encapsulin

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Supramolecule #1: Encapsulin

SupramoleculeName: Encapsulin / 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: 1.7 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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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: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 18.0 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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