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- PDB-7odw: Model of Haliangium ochraceum encapsulin from icosahedral single ... -

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Basic information

Entry
Database: PDB / ID: 7odw
TitleModel of Haliangium ochraceum encapsulin from icosahedral single particle reconstruction
ComponentsLinocin_M18 bacteriocin protein
KeywordsVIRUS LIKE PARTICLE / Encapsulin / encapsulated ferritin / haliangium ochraceum
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / iron ion transport / intracellular iron ion homeostasis / Type 1 encapsulin shell protein
Function and homology information
Biological speciesHaliangium ochraceum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsMarles-Wright, J. / Basle, A. / Clarke, D.J. / Ross, J.
Funding support United Kingdom, 1items
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: Biorxiv / Year: 2021
Title: Model of Haliangium ochraceum encapsulin from icosahedral single particle reconstruction
Authors: Marles-Wright, J. / Basle, A. / Clarke, D.J. / Ross, J.
History
DepositionApr 30, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 9, 2022Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Linocin_M18 bacteriocin protein


Theoretical massNumber of molelcules
Total (without water)28,8451
Polymers28,8451
Non-polymers00
Water00
1
A: Linocin_M18 bacteriocin protein
x 60


Theoretical massNumber of molelcules
Total (without water)1,730,67660
Polymers1,730,67660
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59

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Components

#1: Protein Linocin_M18 bacteriocin protein


Mass: 28844.598 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Haliangium ochraceum (strain DSM 14365 / JCM 11303 / SMP-2) (bacteria)
Strain: DSM 14365 / JCM 11303 / SMP-2 / Gene: Hoch_3837 / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: D0LZ74

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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: Ternary complex of Haliangium ochraceum encapsulin and encapsulated ferritin proteins
Type: COMPLEX / Details: Complex produced by co-expression in E. coli / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.8 MDa / Experimental value: NO
Source (natural)Organism: Haliangium ochraceum (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Details: Solutions were prepared with MilliQ water and filtered using a 0.22 um filter.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium ChlorideNaCl1
220 mMHEPES1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample mono disperse as determined by SEC
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K
Details: blot force -5, wait time 10 seconds and blot time of 3 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 40.509 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8109
Image scansWidth: 11520 / Height: 8184

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4CTFFIND4CTF correction
5RELION3.1CTF correctionCTFrefine
8UCSF Chimera1.1.1model fitting
10RELION3.1initial Euler assignmentInitial model
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.18model refinement
CTF correctionDetails: CTF correction after motion correction followed by CFT refinement after 3D Refinement.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 340403
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 254134 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 86 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: CC
Details: Initial fitting performed using chimera with real space refinement in Phenix.
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 102.75 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0062070
ELECTRON MICROSCOPYf_angle_d0.712820
ELECTRON MICROSCOPYf_dihedral_angle_d11.513289
ELECTRON MICROSCOPYf_chiral_restr0.046318
ELECTRON MICROSCOPYf_plane_restr0.004375

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