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- EMDB-23834: T4GALA Engineered Protein Nanocage -

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Basic information

Entry
Database: EMDB / ID: EMD-23834
TitleT4GALA Engineered Protein Nanocage
Map data
Sample
  • Complex: T4GALA Engineered Protein Nanocage
    • Protein or peptide: T4GALA Engineered Protein Nanocage
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsAndreas MP / Jones JA / Cristie-David AS / Giessen TW
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseSep 29, 2021-
Map releaseSep 29, 2021-
UpdateNov 24, 2021-
Current statusNov 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mh2
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7mh2
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23834.png

Downloads & links

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Map

FileDownload / File: emd_23834.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.40318 Å
Density
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.45
Minimum - Maximum-0.5600228 - 1.5971866
Average (Standard dev.)0.006595672 (±0.102173164)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 617.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.40318181818181.40318181818181.4031818181818
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z617.400617.400617.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.5601.5970.007

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Supplemental data

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Sample components

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Entire : T4GALA Engineered Protein Nanocage

EntireName: T4GALA Engineered Protein Nanocage
Components
  • Complex: T4GALA Engineered Protein Nanocage
    • Protein or peptide: T4GALA Engineered Protein Nanocage

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Supramolecule #1: T4GALA Engineered Protein Nanocage

SupramoleculeName: T4GALA Engineered Protein Nanocage / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Quasibacillus thermotolerans (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: T4GALA Engineered Protein Nanocage

MacromoleculeName: T4GALA Engineered Protein Nanocage / type: protein_or_peptide / ID: 1
Details: Amino Acids 58-83: synthetic GALA peptide insertion; Amino Acids 305-310: Linker; Amino Acids 311-316: Affinity Tag
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Quasibacillus thermotolerans (bacteria)
Molecular weightTheoretical: 35.290652 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKSQLYPDS PLTDQDFNQL DQTVIEAARR QLVGRRFIEL YGPLGRGMQS VFNDIFMESH EGGGEALAEA LAEALAEALA GGGAKMDFQ GSFDTEVESS RRVNYTIPML YKDFVLYWRD LEQSKALDIP IDFSVAANAA RDVAFLEDQM IFHGSKEFDI P GLMNVKGR ...String:
MNKSQLYPDS PLTDQDFNQL DQTVIEAARR QLVGRRFIEL YGPLGRGMQS VFNDIFMESH EGGGEALAEA LAEALAEALA GGGAKMDFQ GSFDTEVESS RRVNYTIPML YKDFVLYWRD LEQSKALDIP IDFSVAANAA RDVAFLEDQM IFHGSKEFDI P GLMNVKGR LTHLIGNWYE SGNAFQDIVE ARNKLLEMNH NGPYALVLSP ELYSLLHRVH KDTNVLEIEH VRELITAGVF QS PVLKGKS GVIVNTGRNN LDLAISEDFE TAYLGEEGMN HPFRVYETVV LRIKRPAAIC TLIDPEEGGG GGGHHHHHH

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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

Concentration0.76 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC11H26N2O6Bis Tris Propane
150.0 mMNaClSodium chlorideSodium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 60 seconds, 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
Details: Blot force: 20 Blot time: 4 seconds Wait time: 0 seconds.

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -1.8 µm / Nominal defocus min: -1.3 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1259 / Average exposure time: 8.0 sec. / Average electron dose: 62.0 e/Å2

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 2.15.00) / Software - details: Patch CTF Estimation
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6nj8

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.15.00)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2.15.00)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15.00) / Software - details: Homogenous Refinement / Number images used: 6707

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Atomic model buiding 1

Initial modelPDB ID:

6nj8

DetailsModel 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 81.22 / Target criteria: Correlation Coefficient
Output model

PDB-7mh2:
T4GALA Engineered Protein Nanocage

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