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- PDB-9hq7: Partial (52mer) encapsulin shell assembly from Mycobacterium tube... -

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Basic information

Entry
Database: PDB / ID: 9hq7
TitlePartial (52mer) encapsulin shell assembly from Mycobacterium tuberculosis
ComponentsType 1 encapsulin shell protein
KeywordsVIRUS LIKE PARTICLE / encapsulin
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.51 Å
AuthorsLewis, C.J. / Berger, C. / Ravelli, R.B.G.
Funding support Netherlands, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
Other governmentLHSM18067
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
DepositionDec 16, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type 1 encapsulin shell protein
B: Type 1 encapsulin shell protein
D: Type 1 encapsulin shell protein
E: Type 1 encapsulin shell protein
F: Type 1 encapsulin shell protein
G: Type 1 encapsulin shell protein
H: Type 1 encapsulin shell protein
I: Type 1 encapsulin shell protein
K: Type 1 encapsulin shell protein
L: Type 1 encapsulin shell protein
M: Type 1 encapsulin shell protein
N: Type 1 encapsulin shell protein
O: Type 1 encapsulin shell protein
P: Type 1 encapsulin shell protein
Q: Type 1 encapsulin shell protein
R: Type 1 encapsulin shell protein
S: Type 1 encapsulin shell protein
T: Type 1 encapsulin shell protein
U: Type 1 encapsulin shell protein
V: Type 1 encapsulin shell protein
W: Type 1 encapsulin shell protein
X: Type 1 encapsulin shell protein
BA: Type 1 encapsulin shell protein
CA: Type 1 encapsulin shell protein
DA: Type 1 encapsulin shell protein
EA: Type 1 encapsulin shell protein
FA: Type 1 encapsulin shell protein
GA: Type 1 encapsulin shell protein
HA: Type 1 encapsulin shell protein
IA: Type 1 encapsulin shell protein
JA: Type 1 encapsulin shell protein
KA: Type 1 encapsulin shell protein
LA: Type 1 encapsulin shell protein
MA: Type 1 encapsulin shell protein
NA: Type 1 encapsulin shell protein
OA: Type 1 encapsulin shell protein
PA: Type 1 encapsulin shell protein
QA: Type 1 encapsulin shell protein
SA: Type 1 encapsulin shell protein
TA: Type 1 encapsulin shell protein
UA: Type 1 encapsulin shell protein
VA: Type 1 encapsulin shell protein
WA: Type 1 encapsulin shell protein
XA: Type 1 encapsulin shell protein
YA: Type 1 encapsulin shell protein
ZA: Type 1 encapsulin shell protein
AB: Type 1 encapsulin shell protein
CB: Type 1 encapsulin shell protein
DB: Type 1 encapsulin shell protein
FB: Type 1 encapsulin shell protein
GB: Type 1 encapsulin shell protein
HB: Type 1 encapsulin shell protein


Theoretical massNumber of molelcules
Total (without water)1,500,89452
Polymers1,500,89452
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein ...
Type 1 encapsulin shell protein / Culture filtrate protein 29 / CFP29


Mass: 28863.346 Da / Num. of mol.: 52
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: enc, cfp29, Rv0798c / Plasmid: pRSET / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41 / References: UniProt: I6WZG6
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: Partial encapsulin shell from Mycobacterium tuberculosis
Type: ORGANELLE OR CELLULAR COMPONENT / Details: 52 of 60 monomers / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: Rv0798c
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41 / Plasmid: pRSET
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 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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2RELION3.1image acquisition
3EPUimage acquisition
4Gctfimage acquisition
5PHENIX1.18.2image acquisition
6Coot0.9.5image acquisition
8GctfCTF correction
11PHENIX1.18.2model fitting
12Coot0.9.5model fitting
14PHENIX1.18.2model refinement
15Coot0.9.5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 89072
3D reconstructionResolution: 4.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6294 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 7P1T

7p1t


Accession code: 7P1T / Source name: PDB / Type: experimental model

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