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- EMDB-11595: Shotgun EM of Mycobacterial protein complexes during stationary p... -

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Basic information

Entry
Database: EMDB / ID: EMD-11595
TitleShotgun EM of Mycobacterial protein complexes during stationary phase stress.
Map dataAspartyl aminopeptidase from Mycobacterium smegmatis
Sample
  • Complex: Probable M18 family aminopeptidase 2
    • Protein or peptide: Aspartyl aminopeptidase
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 26.0 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: 0.0399
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0399
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11595.png

Downloads & links

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Map

FileDownload / File: emd_11595.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAspartyl aminopeptidase from Mycobacterium smegmatis
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.84 Å/pix.
x 120 pix.
= 460.8 Å		3.84 Å/pix.
x 120 pix.
= 460.8 Å		3.84 Å/pix.
x 120 pix.
= 460.8 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: 0.0399 / Movie #1: 0.0399
Minimum - Maximum-0.04729205 - 0.1612502
Average (Standard dev.)0.00044288335 (±0.00899658)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 460.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.843.843.84
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.0470.1610.000

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Supplemental data

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Sample components

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Entire : Probable M18 family aminopeptidase 2

EntireName: Probable M18 family aminopeptidase 2
Components
  • Complex: Probable M18 family aminopeptidase 2
    • Protein or peptide: Aspartyl aminopeptidase

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Supramolecule #1: Probable M18 family aminopeptidase 2

SupramoleculeName: Probable M18 family aminopeptidase 2 / 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: 540 KDa

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Macromolecule #1: Aspartyl aminopeptidase

MacromoleculeName: Aspartyl aminopeptidase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: groELDC
SequenceString: MAASPHSLCE FIDASPSPFH VCATAAARLR DAGYTELAET DAWPAAGRFF TVRAGSLVAW RTVEDASAPF RIVGGHTDSP NLRVKQRPDR MVAGWQVVAL QPYGGAWLNS WLDRDLGISG RLTLRDESAD DGIAHHLVRI DDPILRVPQL AIHLSDDRKG VSPDPQRHLN ...String:
MAASPHSLCE FIDASPSPFH VCATAAARLR DAGYTELAET DAWPAAGRFF TVRAGSLVAW RTVEDASAPF RIVGGHTDSP NLRVKQRPDR MVAGWQVVAL QPYGGAWLNS WLDRDLGISG RLTLRDESAD DGIAHHLVRI DDPILRVPQL AIHLSDDRKG VSPDPQRHLN GVWGLGERPG VFIEFVADRA GVDAADVLGF DLMTHDLAPS AVTGAAGEFV SAPRLDNQAT CYAGLEAFLA AEESGYLPVL ALFDHEEVGS QSDHGAQSEL LPTVLERIAL AAGQSREDFL RRVAGSMVAS GDMAHATHPN YPERHEPGHL IEVNAGPVLK VQPNLRYATD GRTAAAFALA CDQAGVPLQR YEHRADLPCG STIGPMTAAR TGIPTVDVGA AQLAMHSARE FMGAHDVAAY SAALQAFLSP A

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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 / Material: COPPER / Mesh: 400 / 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: 200 / 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: 49751
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: 3 / Applied symmetry - Point group: T (tetrahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 26.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 1928
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: 200 / Avg.num./class: 250 / 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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