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- EMDB-13608: Focused reconstruction of Haliangium ochraceum encapsulated ferri... -

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Basic information

Entry
Database: EMDB / ID: EMD-13608
TitleFocused reconstruction of Haliangium ochraceum encapsulated ferritin cargo within the encapsulin nano compartment
Map dataCryroSPARC map
Sample
  • Complex: Ternary complex of Haliangium ochraceum encapsulin and encapsulated ferritin proteins
    • Complex: Encapsulin nano compartment
      • Protein or peptide: Haliangium ochraceum encapsulin
      • Protein or peptide: Haliangium ochraceum encapsulated ferritin
Biological speciesHaliangium ochraceum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.5 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
CitationJournal: 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.
History
DepositionSep 21, 2021-
Header (metadata) releaseAug 3, 2022-
Map releaseAug 3, 2022-
UpdateAug 3, 2022-
Current statusAug 3, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13608.png

Downloads & links

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Map

FileDownload / File: emd_13608.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryroSPARC map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 480 pix.
= 312.96 Å		0.65 Å/pix.
x 480 pix.
= 312.96 Å		0.65 Å/pix.
x 480 pix.
= 312.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.652 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.07714641 - 0.2058275
Average (Standard dev.)0.0004760953 (±0.0051814537)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_13608_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map

Fileemd_13608_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_13608_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_13608_half_map_2.map
AnnotationHalf map B
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
    • Complex: Encapsulin nano compartment
      • 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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Supramolecule #2: Encapsulin nano compartment

SupramoleculeName: Encapsulin nano compartment / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Haliangium ochraceum (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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 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
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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 335921
CTF correctionSoftware: (Name: CTFFIND (ver. 4), cryoSPARC (ver. 3.2))
Details: CTF correction after motion correction followed by CFT refinement after 3D Refinement.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2)
Details: Homogeneous masked 3D refinementment in CryoSPARC on D2 symmetry expanded particle set
Number images used: 13335780
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2) / Details: Five initial models produced in CryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 5 / Avg.num./class: 60000 / Software - Name: RELION (ver. 3.1) / Details: Heterogeneous refinement of five ab initio models
FSC plot (resolution estimation)

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