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

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Basic information

Entry
Database: PDB / ID: 7p1t
TitleCryo-EM structure of encapsulin from Mycobacterium tuberculosis
Components29 kDa antigen, Cfp29
KeywordsVIRUS LIKE PARTICLE / encapsulin / cfp29 / nanocompartment
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / : / encapsulin nanocompartment / hydrolase activity / plasma membrane / Type 1 encapsulin shell protein
Function and homology information
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.29 Å
AuthorsLewis, C.J. / Berger, C. / Ravelli, R.B.G.
Funding support Netherlands, 2items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
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Revision 1.1Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update
Revision 1.2Nov 13, 2024Group: Data collection / Structure summary / Category: em_admin / pdbx_entry_details / Item: _em_admin.last_update
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 29 kDa antigen, Cfp29
B: 29 kDa antigen, Cfp29
C: 29 kDa antigen, Cfp29
D: 29 kDa antigen, Cfp29
E: 29 kDa antigen, Cfp29
F: 29 kDa antigen, Cfp29
G: 29 kDa antigen, Cfp29
H: 29 kDa antigen, Cfp29
I: 29 kDa antigen, Cfp29
J: 29 kDa antigen, Cfp29
K: 29 kDa antigen, Cfp29
L: 29 kDa antigen, Cfp29
M: 29 kDa antigen, Cfp29
N: 29 kDa antigen, Cfp29
O: 29 kDa antigen, Cfp29
P: 29 kDa antigen, Cfp29
Q: 29 kDa antigen, Cfp29
R: 29 kDa antigen, Cfp29
S: 29 kDa antigen, Cfp29
T: 29 kDa antigen, Cfp29
U: 29 kDa antigen, Cfp29
V: 29 kDa antigen, Cfp29
W: 29 kDa antigen, Cfp29
X: 29 kDa antigen, Cfp29
Y: 29 kDa antigen, Cfp29
Z: 29 kDa antigen, Cfp29
AA: 29 kDa antigen, Cfp29
BA: 29 kDa antigen, Cfp29
CA: 29 kDa antigen, Cfp29
DA: 29 kDa antigen, Cfp29
EA: 29 kDa antigen, Cfp29
FA: 29 kDa antigen, Cfp29
GA: 29 kDa antigen, Cfp29
HA: 29 kDa antigen, Cfp29
IA: 29 kDa antigen, Cfp29
JA: 29 kDa antigen, Cfp29
KA: 29 kDa antigen, Cfp29
LA: 29 kDa antigen, Cfp29
MA: 29 kDa antigen, Cfp29
NA: 29 kDa antigen, Cfp29
OA: 29 kDa antigen, Cfp29
PA: 29 kDa antigen, Cfp29
QA: 29 kDa antigen, Cfp29
RA: 29 kDa antigen, Cfp29
SA: 29 kDa antigen, Cfp29
TA: 29 kDa antigen, Cfp29
UA: 29 kDa antigen, Cfp29
VA: 29 kDa antigen, Cfp29
WA: 29 kDa antigen, Cfp29
XA: 29 kDa antigen, Cfp29
YA: 29 kDa antigen, Cfp29
ZA: 29 kDa antigen, Cfp29
AB: 29 kDa antigen, Cfp29
BB: 29 kDa antigen, Cfp29
CB: 29 kDa antigen, Cfp29
DB: 29 kDa antigen, Cfp29
EB: 29 kDa antigen, Cfp29
FB: 29 kDa antigen, Cfp29
GB: 29 kDa antigen, Cfp29
HB: 29 kDa antigen, Cfp29


Theoretical massNumber of molelcules
Total (without water)1,731,80160
Polymers1,731,80160
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area261100 Å2
ΔGint-450 kcal/mol
Surface area567590 Å2
MethodPISA

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Components

#1: Protein ...
29 kDa antigen, Cfp29 / Bacteriocin / Linocin-M18


Mass: 28863.346 Da / Num. of mol.: 60
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: cfp29, lin, C0094_04325, CAB90_00906, DSI38_23045, E5M05_20280, E5M52_19875, E5M78_19835, ERS007661_02029, ERS007665_01536, ERS007679_03499, ERS007688_03248, ERS007741_01530, ERS013471_03708, ...Gene: cfp29, lin, C0094_04325, CAB90_00906, DSI38_23045, E5M05_20280, E5M52_19875, E5M78_19835, ERS007661_02029, ERS007665_01536, ERS007679_03499, ERS007688_03248, ERS007741_01530, ERS013471_03708, ERS023446_03804, ERS027659_01280, ERS094182_03983, F6W99_03480, FRD82_02930, GCL30_20955, SAMEA2683035_02949
Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: A0A045HTX8, Hydrolases; Acting on peptide bonds (peptidases)
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: encapsulin shell from Mycobacterium tuberculosis / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.729 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41
Buffer solutionpH: 7
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 163000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4GctfCTF correction
7PHENIX1.18.2model fitting
8Coot0.9.5model fitting
10PHENIX1.18.2model refinement
11Coot0.9.5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 89072
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57805 / Symmetry type: POINT
Atomic model buildingB value: 34.36 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: multiple
Atomic model buildingPDB-ID: 619G

619g


Accession code: 619G / Source name: PDB / Type: experimental model

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