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- EMDB-12873: Asymmetric single particle reconstruction of the Haliangium ochra... -

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Basic information

Entry
Database: EMDB / ID: EMD-12873
TitleAsymmetric single particle reconstruction of the Haliangium ochraceum encapsulin encapsulated ferritin complex
Map dataSolvent flattened B-factor sharpened map from post process
Sample
  • Complex: Ternary complex of Haliangium ochraceum encapsulin and encapsulated ferritin proteins
    • Protein or peptide: Haliangium ochraceum encapsulin
    • Protein or peptide: Haliangium ochraceum encapsulated ferritin
Function / homology
Function and homology information


encapsulin nanocompartment / ferroxidase / ferroxidase activity / iron ion transport / intracellular iron ion homeostasis / metal ion binding
Similarity search - Function
Ferritin-like protein / Type 1 encapsulin shell protein / Encapsulating protein for peroxidase / Ferritin-like superfamily
Similarity search - Domain/homology
Encapsulated ferritin-like protein / Type 1 encapsulin shell protein
Similarity search - Component
Biological speciesHaliangium ochraceum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMarles-Wright J / Basle A / Ross J / Clarke DJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N005570/1 United Kingdom
Citation
Journal: Sci Adv / Year: 2022
Title: Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment.
Authors: Jennifer Ross / Zak McIver / Thomas Lambert / Cecilia Piergentili / Jasmine Emma Bird / Kelly J Gallagher / Faye L Cruickshank / Patrick James / Efrain Zarazúa-Arvizu / Louise E Horsfall / ...Authors: Jennifer Ross / Zak McIver / Thomas Lambert / Cecilia Piergentili / Jasmine Emma Bird / Kelly J Gallagher / Faye L Cruickshank / Patrick James / Efrain Zarazúa-Arvizu / Louise E Horsfall / Kevin J Waldron / Marcus D Wilson / C Logan Mackay / Arnaud Baslé / David J Clarke / Jon Marles-Wright /
Abstract: Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant encapsulin:encapsulated ferritin ...Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant encapsulin:encapsulated ferritin complex using cryo-electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.
#1: Journal: To Be Published
Title: Model of Haliangium ochraceum encapsulin from icosahedral single particle reconstruction
Authors: Marles-Wright J / Basle A / Clarke DJ / Ross J
History
DepositionMay 4, 2021-
Header (metadata) releaseFeb 9, 2022-
Map releaseFeb 9, 2022-
UpdateFeb 9, 2022-
Current statusFeb 9, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.011
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.011
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12873.png

Downloads & links

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Map

FileDownload / File: emd_12873.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSolvent flattened B-factor sharpened map from post process
Voxel sizeX=Y=Z: 0.652 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.011
Minimum - Maximum-0.034663692 - 0.05973461
Average (Standard dev.)0.0006200315 (±0.0033476667)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 312.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6520.6520.652
M x/y/z480480480
origin x/y/z0.0000.0000.000
length x/y/z312.960312.960312.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS480480480
D min/max/mean-0.0350.0600.001

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Supplemental data

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Mask #1

Fileemd_12873_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Refine 3D map

Fileemd_12873_additional_1.map
AnnotationRefine 3D map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_12873_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_12873_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of Haliangium ochraceum encapsulin and encapsulat...

EntireName: Ternary complex of Haliangium ochraceum encapsulin and encapsulated ferritin proteins
Components
  • Complex: Ternary complex of Haliangium ochraceum encapsulin and encapsulated ferritin proteins
    • Protein or peptide: Haliangium ochraceum encapsulin
    • Protein or peptide: Haliangium ochraceum encapsulated ferritin

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Supramolecule #1: Ternary complex of Haliangium ochraceum encapsulin and encapsulat...

SupramoleculeName: Ternary complex of Haliangium ochraceum encapsulin and encapsulated ferritin proteins
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Complex produced by co-expression in E. coli
Source (natural)Organism: Haliangium ochraceum (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 1.8 MDa

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Macromolecule #1: Haliangium ochraceum encapsulin

MacromoleculeName: Haliangium ochraceum encapsulin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Haliangium ochraceum (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDLLKRHLAP IVPDAWSAID EEAKEIFQGH LAGRKLVDFR GPFGWEYAAV NTGELRPIDD TPEDVDMKL RQVQPLAEVR VPFTLDVTEL DSVARGATNP DLDDVARAAE RMVEAEDSAI F HGWAQAGI KGIVDSTPHE ALAVASVSDF PRAVLSAADT LRKAGVTGPY ...String:
MDLLKRHLAP IVPDAWSAID EEAKEIFQGH LAGRKLVDFR GPFGWEYAAV NTGELRPIDD TPEDVDMKL RQVQPLAEVR VPFTLDVTEL DSVARGATNP DLDDVARAAE RMVEAEDSAI F HGWAQAGI KGIVDSTPHE ALAVASVSDF PRAVLSAADT LRKAGVTGPY ALVLGPKAYD DL FAATQDG YPVAKQVQRL VVDGPLVRAN ALAGALVMSM RGGDYELTVG QDLSIGYAFH DRS KVELFV AESFTFRVLE PGAAVHLRYA

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Macromolecule #2: Haliangium ochraceum encapsulated ferritin

MacromoleculeName: Haliangium ochraceum encapsulated ferritin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Haliangium ochraceum (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSSEQLHEPA ELLSEETKNM HRALVTLIEE LEAVDWYQQR ADACSEPGLH DVLIHNKNEE VEHAMMTLE WIRRRSPVFD AHMRTYLFTE RPILELEEED TGSSSSVAAS PTSAPSHGSL G IGSLRQEG KED

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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

Concentration3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium Chloride
20.0 mMHEPES

Details: Solutions were prepared with MilliQ water and filtered using a 0.22 um filter.
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV
Details: blot force -5, wait time 10 seconds and blot time of 3 seconds.
DetailsSample mono disperse as determined by SEC

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 8109 / Average exposure time: 1.0 sec. / Average electron dose: 40.509 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 340403
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4)
RELION (ver. 3.1)CTFrefine

Details: CTF correction after motion correction followed by CFT refinement after 3D Refinement.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Software - details: Initial model / Details: Initial model produced in Relion
Final 3D classificationNumber classes: 3 / Avg.num./class: 254134 / Software - Name: RELION (ver. 3.1)
Details: Final 3D classification used to remove broken particles.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1) / Software - details: Refine 3D
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 68096
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsInitial fitting performed using chimera with real space refinement in Phenix.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 86 / Target criteria: CC

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