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- EMDB-4443: Structure of left-handed protein cage consisting of 24 eleven-mem... -

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Basic information

Entry
Database: EMDB / ID: EMD-4443
TitleStructure of left-handed protein cage consisting of 24 eleven-membered ring proteins held together by gold (I) bridges.
Map datamap of the left-handed protein cage
Sample
  • Complex: Designed protein cage consisting of C11-symmetric TRAP proteins coordinated by Au+1 ionsDesign
    • Protein or peptide: Transcription attenuation protein MtrB
  • Ligand: GOLD ION
Function / homologyTranscription attenuation protein MtrB / Tryptophan RNA-binding attenuator protein domain / Tryptophan RNA-binding attenuator protein / Tryptophan RNA-binding attenuator protein-like domain superfamily / DNA-templated transcription termination / regulation of DNA-templated transcription / RNA binding / identical protein binding / Transcription attenuation protein MtrB
Function and homology information
Biological speciesGeobacillus stearothermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsMalay AD / Miyazaki N / Biela AP / Iwasaki K / Heddle JG
CitationJournal: Nature / Year: 2019
Title: An ultra-stable gold-coordinated protein cage displaying reversible assembly.
Authors: Ali D Malay / Naoyuki Miyazaki / Artur Biela / Soumyananda Chakraborti / Karolina Majsterkiewicz / Izabela Stupka / Craig S Kaplan / Agnieszka Kowalczyk / Bernard M A G Piette / Georg K A ...Authors: Ali D Malay / Naoyuki Miyazaki / Artur Biela / Soumyananda Chakraborti / Karolina Majsterkiewicz / Izabela Stupka / Craig S Kaplan / Agnieszka Kowalczyk / Bernard M A G Piette / Georg K A Hochberg / Di Wu / Tomasz P Wrobel / Adam Fineberg / Manish S Kushwah / Mitja Kelemen / Primož Vavpetič / Primož Pelicon / Philipp Kukura / Justin L P Benesch / Kenji Iwasaki / Jonathan G Heddle /
Abstract: Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery, and have inspired synthetic biologists to create novel protein assemblies ...Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Au-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
History
DepositionNov 28, 2018-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 6, 2019-
UpdateJun 12, 2019-
Current statusJun 12, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.115
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.115
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rvv
  • Surface level: 0.115
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6rvv
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4443.png

Downloads & links

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Map

FileDownload / File: emd_4443.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of the left-handed protein cage
Voxel sizeX=Y=Z: 1.74 Å
Density
Contour LevelBy AUTHOR: 0.115 / Movie #1: 0.115
Minimum - Maximum-0.20715603 - 0.4576644
Average (Standard dev.)0.0025821018 (±0.02532443)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 382.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.741.741.74
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z382.800382.800382.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.2070.4580.003

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Supplemental data

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Sample components

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Entire : Designed protein cage consisting of C11-symmetric TRAP proteins c...

EntireName: Designed protein cage consisting of C11-symmetric TRAP proteins coordinated by Au+1 ionsDesign
Components
  • Complex: Designed protein cage consisting of C11-symmetric TRAP proteins coordinated by Au+1 ionsDesign
    • Protein or peptide: Transcription attenuation protein MtrB
  • Ligand: GOLD ION

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Supramolecule #1: Designed protein cage consisting of C11-symmetric TRAP proteins c...

SupramoleculeName: Designed protein cage consisting of C11-symmetric TRAP proteins coordinated by Au+1 ions
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET21b
Molecular weightExperimental: 2.2 MDa

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Macromolecule #1: Transcription attenuation protein MtrB

MacromoleculeName: Transcription attenuation protein MtrB / type: protein_or_peptide / ID: 1 / Number of copies: 264 / Enantiomer: LEVO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 8.161224 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MYTNSDFVVI KALEDGVNVI GLTRGADTRF HHSECLDKGE VLIAQFTEHT SAIKVRGKAY IQTSHGVIES EGKK

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Macromolecule #2: GOLD ION

MacromoleculeName: GOLD ION / type: ligand / ID: 2 / Number of copies: 120 / Formula: AU
Molecular weightTheoretical: 196.967 Da

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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

Concentration0.89 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3.0 s blotting time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: An Initial model was produced by EMAN1.9 using the images acquired using JEM2200FS(JEOL,Tokyo) equipped with K2 Summit. The model was refined using RELION up to 16.5A. This was used as a ...Details: An Initial model was produced by EMAN1.9 using the images acquired using JEM2200FS(JEOL,Tokyo) equipped with K2 Summit. The model was refined using RELION up to 16.5A. This was used as a reference model to start RELION in this experiment.
Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 176463
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4v4f

chain_id: AA

4v4f

chain_id: AB

4v4f

chain_id: AC

4v4f

chain_id: AD

4v4f

chain_id: AE

4v4f

chain_id: AF

4v4f

chain_id: AG

4v4f

chain_id: AH

4v4f

chain_id: AI

4v4f

chain_id: AJ

4v4f

chain_id: AK
RefinementSpace: REAL / Protocol: OTHER
Target criteria: gradient-driven minimization of combined map and restraints target
Output model

PDB-6rvv:
Structure of left-handed protein cage consisting of 24 eleven-membered ring proteins held together by gold (I) bridges.

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