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- PDB-7mh2: T4GALA Engineered Protein Nanocage -

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Basic information

Entry
Database: PDB / ID: 7mh2
TitleT4GALA Engineered Protein Nanocage
ComponentsT4GALA Engineered Protein Nanocage
KeywordsVIRUS LIKE PARTICLE / Encapsulin / Nanocage / Nanocompartment
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 speciesQuasibacillus thermotolerans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsAndreas, M.P. / Jones, J.A. / Cristie-David, A.S. / Giessen, T.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R35GM133325 United States
CitationJournal: Angew Chem Int Ed Engl / Year: 2021
Title: Triggered Reversible Disassembly of an Engineered Protein Nanocage*.
Authors: Jesse A Jones / Ajitha S Cristie-David / Michael P Andreas / Tobias W Giessen /
Abstract: Protein nanocages play crucial roles in sub-cellular compartmentalization and spatial control in all domains of life and have been used as biomolecular tools for applications in biocatalysis, drug ...Protein nanocages play crucial roles in sub-cellular compartmentalization and spatial control in all domains of life and have been used as biomolecular tools for applications in biocatalysis, drug delivery, and bionanotechnology. The ability to control their assembly state under physiological conditions would further expand their practical utility. To gain such control, we introduced a peptide capable of triggering conformational change at a key structural position in the largest known encapsulin nanocompartment. We report the structure of the resulting engineered nanocage and demonstrate its ability to disassemble and reassemble on demand under physiological conditions. We demonstrate its capacity for in vivo encapsulation of proteins of choice while also demonstrating in vitro cargo loading capabilities. Our results represent a functionally robust addition to the nanocage toolbox and a novel approach for controlling protein nanocage disassembly and reassembly under mild conditions.
History
DepositionApr 14, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 29, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.name
Revision 1.2May 29, 2024Group: Data collection / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-23834
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-23834
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: T4GALA Engineered Protein Nanocage
B: T4GALA Engineered Protein Nanocage
C: T4GALA Engineered Protein Nanocage
D: T4GALA Engineered Protein Nanocage


Theoretical massNumber of molelcules
Total (without water)141,1634
Polymers141,1634
Non-polymers00
Water0
1
A: T4GALA Engineered Protein Nanocage
B: T4GALA Engineered Protein Nanocage
C: T4GALA Engineered Protein Nanocage
D: T4GALA Engineered Protein Nanocage
x 60


Theoretical massNumber of molelcules
Total (without water)8,469,756240
Polymers8,469,756240
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: T4GALA Engineered Protein Nanocage
B: T4GALA Engineered Protein Nanocage
C: T4GALA Engineered Protein Nanocage
D: T4GALA Engineered Protein Nanocage
x 5


  • icosahedral pentamer
  • 706 kDa, 20 polymers
Theoretical massNumber of molelcules
Total (without water)705,81320
Polymers705,81320
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: T4GALA Engineered Protein Nanocage
B: T4GALA Engineered Protein Nanocage
C: T4GALA Engineered Protein Nanocage
D: T4GALA Engineered Protein Nanocage
x 6


  • icosahedral 23 hexamer
  • 847 kDa, 24 polymers
Theoretical massNumber of molelcules
Total (without water)846,97624
Polymers846,97624
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
T4GALA Engineered Protein Nanocage


Mass: 35290.652 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Amino Acids 58-83: synthetic GALA peptide insertion; Amino Acids 305-310: Linker; Amino Acids 311-316: Affinity Tag
Source: (gene. exp.) Quasibacillus thermotolerans (bacteria)
Gene: QY95_01592 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0F5HPP7

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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: T4GALA Engineered Protein Nanocage / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Quasibacillus thermotolerans (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMBis Tris PropaneC11H26N2O61
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 0.76 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 60 seconds, 5 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: Blot force: 20 Blot time: 4 seconds Wait time: 0 seconds

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: -1800 nm / Nominal defocus min: -1300 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 62 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1259
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC2.15.00particle selectionManual Picker
2cryoSPARC2.15.00particle selectionTemplate Picker
3Leginonimage acquisition
5cryoSPARC2.15.00CTF correctionPatch CTF Estimation
8UCSF Chimera1.14model fitting
9Coot0.9model fitting
11cryoSPARC2.15.00initial Euler assignment
12cryoSPARC2.15.00final Euler assignment
14cryoSPARC2.15.003D reconstructionHomogenous Refinement
15PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6707 / Symmetry type: POINT
Atomic model buildingB value: 81.22 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Details: Model was initially docked in Chimera using Fit to Map command. Chains were manually refined in Coot with rigid body refinement, chain refinement, and iterative real space refinements. ASU ...Details: Model was initially docked in Chimera using Fit to Map command. Chains were manually refined in Coot with rigid body refinement, chain refinement, and iterative real space refinements. ASU was then refined using phenix.real_space_refine with default parameters. NCS operators were applied and refined again with NCS contstraints, global minimization, and ADP refinement.
Atomic model buildingPDB-ID: 6NJ8

6nj8


Accession code: 6NJ8 / Source name: PDB / Type: experimental model

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