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- PDB-7lik: Thermotoga maritima Encapsulin Nanocompartment Pore Mutant S1R -

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Basic information

Entry
Database: PDB / ID: 7lik
TitleThermotoga maritima Encapsulin Nanocompartment Pore Mutant S1R
ComponentsMaritimacin
KeywordsVIRUS LIKE PARTICLE / Encapsulin / 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
RIBOFLAVIN / Type 1 encapsulin shell protein
Similarity search - Component
Biological speciesThermotoga maritima (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.91 Å
AuthorsAndreas, M.P. / Adamson, L. / Tasneem, N. / Close, W. / Giessen, T. / Lau, Y.H.
Funding support Australia, United States, 3items
OrganizationGrant numberCountry
Australian Research Council (ARC)DE190100624 Australia
Other privateWRF2020 Australia
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM133325 United States
CitationJournal: Sci Adv / Year: 2022
Title: Pore structure controls stability and molecular flux in engineered protein cages.
Authors: Lachlan S R Adamson / Nuren Tasneem / Michael P Andreas / William Close / Eric N Jenner / Taylor N Szyszka / Reginald Young / Li Chen Cheah / Alexander Norman / Hugo I MacDermott-Opeskin / ...Authors: Lachlan S R Adamson / Nuren Tasneem / Michael P Andreas / William Close / Eric N Jenner / Taylor N Szyszka / Reginald Young / Li Chen Cheah / Alexander Norman / Hugo I MacDermott-Opeskin / Megan L O'Mara / Frank Sainsbury / Tobias W Giessen / Yu Heng Lau /
Abstract: Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of ...Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.
History
DepositionJan 27, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _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.year
Revision 1.2May 29, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / 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

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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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-23381
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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)30,9122
Polymers30,5361
Non-polymers3761
Water00
1
A: Maritimacin
hetero molecules
x 60


Theoretical massNumber of molelcules
Total (without water)1,854,732120
Polymers1,832,15060
Non-polymers22,58260
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
  • 155 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)154,56110
Polymers152,6795
Non-polymers1,8825
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)185,47312
Polymers183,2156
Non-polymers2,2586
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 / Encapsulin Nanocompartment Pore


Mass: 30535.834 Da / Num. of mol.: 1 / Mutation: H187R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (bacteria)
Strain: ATCC 43589 / MSB8 / DSM 3109 / JCM 10099 / Gene: TM_0785 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9WZP2, Hydrolases; Acting on peptide bonds (peptidases)
#2: Chemical ChemComp-RBF / RIBOFLAVIN / RIBOFLAVINE / VITAMIN B2


Mass: 376.364 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H20N4O6
Has ligand of interestN

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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: Thermotoga maratima Pore Mutant S1R / Type: COMPLEX
Details: Thermotoga maratima encapsulin pore mutant with H187R mutation
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.852395 MDa / Experimental value: NO
Source (natural)Organism: Thermotoga maritima MSB8 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
225 mMTrisC4H11NO31
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 60 seconds at 5 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: blot 4 seconds, force 20 immediately prior to plunging into ethane

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: -1800 nm / Nominal defocus min: -1300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 62 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.15.0particle selection
2Leginonimage acquisition
4cryoSPARC2.15.0CTF correction
7UCSF Chimeramodel fitting
8Coot0.9model fitting
10cryoSPARC2.15.0initial Euler assignment
11cryoSPARC2.15.0final Euler assignment
13cryoSPARC2.15.03D reconstruction
14PHENIX1.18.2-3874-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.91 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75829 / Symmetry type: POINT
Atomic model buildingB value: 27.84 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3DKT

3dkt


Pdb chain-ID: A / Accession code: 3DKT / Pdb chain residue range: 1-265 / Source name: PDB / Type: experimental model

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