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- EMDB-13164: Cryo-EM structure of encapsulin from Mycobacterium tuberculosis -

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Basic information

Entry
Database: EMDB / ID: EMD-13164
TitleCryo-EM structure of encapsulin from Mycobacterium tuberculosis
Map dataLocSpiral map
Sample
  • Organelle or cellular component: encapsulin shell from Mycobacterium tuberculosis
    • Protein or peptide: 29 kDa antigen, Cfp29
Keywordsencapsulin / cfp29 / nanocompartment / VIRUS LIKE PARTICLE
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / : / encapsulin nanocompartment / Hydrolases; Acting on peptide bonds (peptidases) / hydrolase activity / 29 kDa antigen, Cfp29
Function and homology information
Biological speciesMycobacterium tuberculosis H37Rv (bacteria) / Mycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.29 Å
AuthorsLewis CJ / Berger C
Funding support Netherlands, 2 items
OrganizationGrant numberCountry
Other governmentLHSM18067 Netherlands
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
CitationJournal: Commun Biol / Year: 2025
Title: In situ and in vitro cryo-EM reveal structures of mycobacterial encapsulin assembly intermediates.
Authors: Casper Berger / Chris Lewis / Ye Gao / Kèvin Knoops / Carmen López-Iglesias / Peter J Peters / Raimond B G Ravelli /
Abstract: Prokaryotes rely on proteinaceous compartments such as encapsulin to isolate harmful reactions. Encapsulin are widely expressed by bacteria, including the Mycobacteriaceae, which include the human ...Prokaryotes rely on proteinaceous compartments such as encapsulin to isolate harmful reactions. Encapsulin are widely expressed by bacteria, including the Mycobacteriaceae, which include the human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Structures of fully assembled encapsulin shells have been determined for several species, but encapsulin assembly and cargo encapsulation are still poorly characterised, because of the absence of encapsulin structures in intermediate assembly states. We combine in situ and in vitro structural electron microscopy to show that encapsulins are dynamic assemblies with intermediate states of cargo encapsulation and shell assembly. Using cryo-focused ion beam (FIB) lamella preparation and cryo-electron tomography (CET), we directly visualise encapsulins in Mycobacterium marinum, and observed ribbon-like attachments to the shell, encapsulin shells with and without cargoes, and encapsulin shells in partially assembled states. In vitro cryo-electron microscopy (EM) single-particle analysis of the Mycobacterium tuberculosis encapsulin was used to obtain three structures of the encapsulin shell in intermediate states, as well as a 2.3 Å structure of the fully assembled shell. Based on the analysis of the intermediate encapsulin shell structures, we propose a model of encapsulin self-assembly via the pairwise addition of monomers.
History
DepositionJul 2, 2021-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateFeb 26, 2025-
Current statusFeb 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13164.png

Downloads & links

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Map

FileDownload / File: emd_13164.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocSpiral map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 320.256 Å		0.83 Å/pix.
x 384 pix.
= 320.256 Å		0.83 Å/pix.
x 384 pix.
= 320.256 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

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Histogram (log scale)
Contour LevelBy AUTHOR: 4.8
Minimum - Maximum0.0 - 29.838861000000001
Average (Standard dev.)0.43837747 (±1.7970632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 320.25598 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13164_msk_1.map
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Additional map: relion postprocess masked

Fileemd_13164_additional_1.map
Annotationrelion postprocess masked
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Half map: halfmap

Fileemd_13164_half_map_1.map
Annotationhalfmap
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Half map: halfmap

Fileemd_13164_half_map_2.map
Annotationhalfmap
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Sample components

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Entire : encapsulin shell from Mycobacterium tuberculosis

EntireName: encapsulin shell from Mycobacterium tuberculosis
Components
  • Organelle or cellular component: encapsulin shell from Mycobacterium tuberculosis
    • Protein or peptide: 29 kDa antigen, Cfp29

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Supramolecule #1: encapsulin shell from Mycobacterium tuberculosis

SupramoleculeName: encapsulin shell from Mycobacterium tuberculosis / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 1.729 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 / EC number: Hydrolases; Acting on peptide bonds (peptidases)
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Molecular weightTheoretical: 28.863346 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 EASVALSH

UniProtKB: 29 kDa antigen, Cfp29

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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

Concentration2.5 mg/mL
BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 1.8 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 163000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 89072
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.29 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 57805
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

619g


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 34.36 / Target criteria: multiple
Output model

PDB-7p1t:
Cryo-EM structure of encapsulin from Mycobacterium tuberculosis

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