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- EMDB-13420: Cryo-EM structure of Mycobacterium tuberculosis encapsulin -

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Basic information

Entry
Database: EMDB / ID: EMD-13420
TitleCryo-EM structure of Mycobacterium tuberculosis encapsulin
Map data
Sample
  • Complex: Empty encapsulin nanocompartment
    • Protein or peptide: 29 kDa antigen CFP29
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / extracellular region / plasma membrane / Type 1 encapsulin shell protein
Function and homology information
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsWoodward JD
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Global Challenges Research FundST/R002754/1 United Kingdom
CitationJournal: To Be Published
Title: Cryo-EM structure of Mycobacterium tuberculosis encapsulin
Authors: Woodward JD
History
DepositionAug 17, 2021-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateJul 20, 2022-
Current statusJul 20, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13420.png

Downloads & links

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Map

FileDownload / File: emd_13420.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.038
Minimum - Maximum-0.107888415 - 0.18769059
Average (Standard dev.)0.000107754095 (±0.0072793486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 457.91998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Empty encapsulin nanocompartment

EntireName: Empty encapsulin nanocompartment
Components
  • Complex: Empty encapsulin nanocompartment
    • Protein or peptide: 29 kDa antigen CFP29

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Supramolecule #1: Empty encapsulin nanocompartment

SupramoleculeName: Empty encapsulin nanocompartment / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET-28a(+)
Molecular weightTheoretical: 1.73 MDa

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Macromolecule #1: 29 kDa antigen CFP29

MacromoleculeName: 29 kDa antigen CFP29 / type: protein_or_peptide / ID: 1 / Number of copies: 60 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 29.554072 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNNLYRDLAP VTEAAWAEIE LEAARTFKRH IAGRRVVDVS DPGGPVTAAV STGRLIDVKA PTNGVIAHLR ASKPLVRLRV PFTLSRNEI DDVERGSKDS DWEPVKEAAK KLAFVEDRTI FEGYSAASIE GIRSASSNPA LTLPEDPREI PDVISQALSE L RLAGVDGP ...String:
MNNLYRDLAP VTEAAWAEIE LEAARTFKRH IAGRRVVDVS DPGGPVTAAV STGRLIDVKA PTNGVIAHLR ASKPLVRLRV PFTLSRNEI DDVERGSKDS DWEPVKEAAK KLAFVEDRTI FEGYSAASIE GIRSASSNPA LTLPEDPREI PDVISQALSE L RLAGVDGP YSVLLSADVY TKVSETSDHG YPIREHLNRL VDGDIIWAPA IDGAFVLTTR GGDFDLQLGT DVAIGYASHD TD TVRLYLQ ETLTFLCYTA EASVALSHHH HHH

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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

Concentration1.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
175.0 mMNaClSodium chloridesodium chloride
25.0 mMC4H11NO3TRIS
250.0 mMC3H4N2imidazol
5.0 % (v/v)C3H8O3glycerol

Details: Eluted in: 50mM Tris-HCl, 350mM NaCl, 10mM Imidazol, 10% v/v glycerol at pH 7.4 and diluted 1:2 in distilled water.
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.5 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.02 kPa
Details: Glow-discharged using an EMS100X in air: 25 mA at 20 Pa for 30 seconds.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsC-flat 2/2 holey carbon film supported by a standard copper TEM grid and coated with an ultrathin (2-3 nm) continuous carbon film.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 14133 / Average electron dose: 45.0 e/Å2
Details: Data collected in super-resolution mode at 0.53 A/pixel and down-sampled during motion correction to 1.06 A/pixel.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2000000
Details: 2D class averages were produced using Relion from 1000 manually-picked particles. These were then used as picking templates.
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER
Details: Initial model generated using Stochastic Gradient Descent.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.1)
Final 3D classificationNumber classes: 50 / Software - Name: RELION (ver. 3.1.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.1)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.1) / Number images used: 1000000
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel was generated ab initio using Buccaneer within the CCPEM package and manually corrected using Coot before being refined using Isolde within ChimeraX and Phenix Real Space Refinement within the CCPEM package.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-7phm:
Cryo-EM structure of Mycobacterium tuberculosis encapsulin

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