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- PDB-7mu1: Thermotoga maritima encapsulin shell -

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Basic information

Entry
Database: PDB / ID: 7mu1
TitleThermotoga maritima encapsulin shell
ComponentsMaritimacin
KeywordsVIRUS LIKE PARTICLE / Encapsulin / Ferritin-like protein / EncFLP / Thermotoga / FMN / Bacterial Nanocompartment
Function / homology
Function and homology information


encapsulin nanocompartment / Hydrolases; Acting on peptide bonds (peptidases) / peptidase activity / iron ion transport / intracellular iron ion homeostasis / proteolysis
Similarity search - Function
Type 1 encapsulin shell protein / Encapsulating protein for peroxidase
Similarity search - Domain/homology
FLAVIN MONONUCLEOTIDE / Type 1 encapsulin shell protein
Similarity search - Component
Biological speciesThermotoga maritima (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLaFrance, B.J. / Nogales, E. / Savage, D.F.
Funding support United States, 2items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-SC00016240 United States
National Science Foundation (NSF, United States)GRFP-1106400 United States
CitationJournal: Sci Rep / Year: 2021
Title: The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein.
Authors: Benjamin J LaFrance / Caleb Cassidy-Amstutz / Robert J Nichols / Luke M Oltrogge / Eva Nogales / David F Savage /
Abstract: Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ...Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.
History
DepositionMay 14, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 24, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 8, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 29, 2024Group: Data collection / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
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 / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
  • Imaged by Jmol
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-24001
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Maritimacin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)30,8262
Polymers30,3701
Non-polymers4561
Water0
1
A: Maritimacin
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)1,849,558120
Polymers1,822,17760
Non-polymers27,38160
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Maritimacin
hetero molecules
x 5


  • icosahedral pentamer
  • 154 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)154,13010
Polymers151,8485
Non-polymers2,2825
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Maritimacin
hetero molecules
x 6


  • icosahedral 23 hexamer
  • 185 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)184,95612
Polymers182,2186
Non-polymers2,7386
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Maritimacin / Thermotoga bacteriocin / T maritima encapsulin shell protein


Mass: 30369.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (bacteria) / Gene: TM_0785 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9WZP2, Hydrolases; Acting on peptide bonds (peptidases)
#2: Chemical ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE / Flavin mononucleotide


Mass: 456.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: 3.3A cryo-EM map of the T. maritima encapsulin shell / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.83 MDa / Experimental value: NO
Source (natural)Organism: Thermotoga maritima (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4
Details: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 5 mM 2-mercaptoethanol
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot force

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2800 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1276
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30

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Processing

SoftwareName: PHENIX / Version: 1.19_4080: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
7PHENIXmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIXmodel refinement
Image processingDetails: Images were saved in counting mode using leginon software, and subsequently processed in RELION.
CTF correctionDetails: CTFFind4 implemented within RELION/3.0, CTFRefine was also performed after initial reconstruction was achieved.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 86124
Details: 1000 particles were manually selected from random micrographs. 2D classification was performed on these 1000 particles, and 2D classes were used for relion auto picking
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38952 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 38 / Protocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 3DKT

3dkt


Pdb chain-ID: A / Accession code: 3DKT / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0032215
ELECTRON MICROSCOPYf_angle_d0.5272995
ELECTRON MICROSCOPYf_dihedral_angle_d5.666294
ELECTRON MICROSCOPYf_chiral_restr0.042329
ELECTRON MICROSCOPYf_plane_restr0.003381

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