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- EMDB-23380: Thermotoga maritima Encapsulin Nanocompartment Pore Mutant S1K -

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Basic information

Entry
Database: EMDB / ID: EMD-23380
TitleThermotoga maritima Encapsulin Nanocompartment Pore Mutant S1K
Map data
Sample
  • Complex: Thermotoga maratima Pore Mutant S1K
    • Protein or peptide: Maritimacin
  • Ligand: RIBOFLAVIN
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / Hydrolases; Acting on peptide bonds (peptidases) / peptidase activity / iron ion transport / intracellular iron ion homeostasis / proteolysis / Type 1 encapsulin shell protein
Function and homology information
Biological speciesThermotoga maritima MSB8 (bacteria) / Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsAndreas MP / Adamson L / Tasneem N / Close W / Giessen T / Lau YH
Funding support Australia, United States, 3 items
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, 2021-
Header (metadata) releaseFeb 9, 2022-
Map releaseFeb 9, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.33
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.33
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lij
  • Surface level: 0.33
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7lij
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23380.png

Downloads & links

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Map

FileDownload / File: emd_23380.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.98 Å
Density
Contour LevelBy AUTHOR: 0.33 / Movie #1: 0.33
Minimum - Maximum-0.955301 - 1.5270555
Average (Standard dev.)0.00542342 (±0.08591775)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 376.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.980.980.98
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z376.320376.320376.320
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.9551.5270.005

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Supplemental data

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Sample components

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Entire : Thermotoga maratima Pore Mutant S1K

EntireName: Thermotoga maratima Pore Mutant S1K
Components
  • Complex: Thermotoga maratima Pore Mutant S1K
    • Protein or peptide: Maritimacin
  • Ligand: RIBOFLAVIN

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Supramolecule #1: Thermotoga maratima Pore Mutant S1K

SupramoleculeName: Thermotoga maratima Pore Mutant S1K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Thermotoga maratima encapsulin pore mutant with H187K mutation
Source (natural)Organism: Thermotoga maritima MSB8 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 1.850714 MDa

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Macromolecule #1: Maritimacin

MacromoleculeName: Maritimacin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: Hydrolases; Acting on peptide bonds (peptidases)
Source (natural)Organism: Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099) (bacteria)
Strain: ATCC 43589 / MSB8 / DSM 3109 / JCM 10099
Molecular weightTheoretical: 30.507818 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEFLKRSFAP LTEKQWQEID NRAREIFKTQ LYGRKFVDVE GPYGWEYAAH PLGEVEVLSD ENEVVKWGLR KSLPLIELRA TFTLDLWEL DNLERGKPNV DLSSLEETVR KVAEFEDEVI FRGCEKSGVK GLLSFEERKI ECGSTPKDLL EAIVRALSIF S KDGIEGPY ...String:
MEFLKRSFAP LTEKQWQEID NRAREIFKTQ LYGRKFVDVE GPYGWEYAAH PLGEVEVLSD ENEVVKWGLR KSLPLIELRA TFTLDLWEL DNLERGKPNV DLSSLEETVR KVAEFEDEVI FRGCEKSGVK GLLSFEERKI ECGSTPKDLL EAIVRALSIF S KDGIEGPY TLVINTDRWI NFLKEEAGKY PLEKRVEECL RGGKIITTPR IEDALVVSER GGDFKLILGQ DLSIGYEDRE KD AVRLFIT ETFTFQVVNP EALILLKF

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Macromolecule #2: RIBOFLAVIN

MacromoleculeName: RIBOFLAVIN / type: ligand / ID: 2 / Number of copies: 1 / Formula: RBF
Molecular weightTheoretical: 376.364 Da
Chemical component information

ChemComp-RBF:
RIBOFLAVIN / Riboflavin

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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

Concentration2.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
25.0 mMC4H11NO3Tris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 60 seconds at 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: blot 4 seconds, force 20 immediately prior to plunging into ethane.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -1.8 µm / Nominal defocus min: -1.3 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average exposure time: 8.0 sec. / Average electron dose: 62.0 e/Å2

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 2.15.0)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3dkt


Details: Initial map for refinements made from PDB entry 3DKT
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.15.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.15.0)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.0) / Number images used: 58162

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Atomic model buiding 1

Initial modelPDB ID:

3dkt


Chain - Chain ID: A / Chain - Residue range: 1-265
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 22.03
Output model

PDB-7lij:
Thermotoga maritima Encapsulin Nanocompartment Pore Mutant S1K

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