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- EMDB-24001: Thermotoga maritima encapsulin shell -

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Basic information

Entry
Database: EMDB / ID: EMD-24001
TitleThermotoga maritima encapsulin shell
Map dataFinal cryo-EM map for the T maritima encapsulin shell
Sample
  • Organelle or cellular component: 3.3A cryo-EM map of the T. maritima encapsulin shell
    • Protein or peptide: Maritimacin
  • Ligand: FLAVIN MONONUCLEOTIDE
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 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLaFrance BJ / Nogales E / Savage DF
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateDec 8, 2021-
Current statusDec 8, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mu1
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mu1
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24001.png

Downloads & links

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Map

FileDownload / File: emd_24001.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal cryo-EM map for the T maritima encapsulin shell
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.035822354 - 0.07585681
Average (Standard dev.)0.0004622815 (±0.003631274)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z419.840419.840419.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ260260260
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0360.0760.000

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Supplemental data

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Mask #1

Fileemd_24001_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 3.3A cryo-EM map of the T. maritima encapsulin shell

EntireName: 3.3A cryo-EM map of the T. maritima encapsulin shell
Components
  • Organelle or cellular component: 3.3A cryo-EM map of the T. maritima encapsulin shell
    • Protein or peptide: Maritimacin
  • Ligand: FLAVIN MONONUCLEOTIDE

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Supramolecule #1: 3.3A cryo-EM map of the T. maritima encapsulin shell

SupramoleculeName: 3.3A cryo-EM map of the T. maritima encapsulin shell / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermotoga maritima (bacteria)
Molecular weightTheoretical: 1.83 MDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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 (bacteria)
Molecular weightTheoretical: 30.369615 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 NFLKEEAGHY PLEKRVEECL RGGKIITTPR IEDALVVSER GGDFKLILGQ DLSIGYEDRE KD AVRLFIT ETFTFQVVNP EALILLK

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Macromolecule #2: FLAVIN MONONUCLEOTIDE

MacromoleculeName: FLAVIN MONONUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 1 / Formula: FMN
Molecular weightTheoretical: 456.344 Da
Chemical component information

ChemComp-FMN:
FLAVIN MONONUCLEOTIDE / Flavin mononucleotide

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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: 7.4
Details: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 5 mM 2-mercaptoethanol
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force.

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Electron microscopy

MicroscopeFEI TITAN
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling 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 / Number grids imaged: 1 / Number real images: 1276 / Average electron dose: 40.0 e/Å2

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Image processing

Particle selectionNumber 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
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Details: CTFFind4 implemented within RELION/3.0, CTFRefine was also performed after initial reconstruction was achieved.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3dkt


Details: Original T. maritima crystal structure from Sutter et al 'molmap' function in Chimera was used to generate surface model. Initial model was lowpass filtered to 30A for initial ...Details: Original T. maritima crystal structure from Sutter et al 'molmap' function in Chimera was used to generate surface model. Initial model was lowpass filtered to 30A for initial reconstruction, and once high-resolution was achieved, a 5A model was used to assure the correct handedness.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 38952
DetailsImages were saved in counting mode using leginon software, and subsequently processed in RELION.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3dkt


Chain - Chain ID: A
RefinementProtocol: FLEXIBLE FIT / Overall B value: 38
Output model

PDB-7mu1:
Thermotoga maritima encapsulin shell

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