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- EMDB-5769: Electron cryo-microscopy of a chemical cross-linked K87C/S199C Mm... -

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Entry
Database: EMDB / ID: EMD-5769
TitleElectron cryo-microscopy of a chemical cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
Map datachemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
Sample
  • Sample: chemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
  • Protein or peptide: mutant Methanococcus maripaludis chaperonin
Keywordsprotein engineering / light-gated nanocage / ABDM cross-linked Mm-cpn
Biological speciesMethanococcus maripaludis (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.4 Å
AuthorsHoersch D / Roh SH / Chiu W / Kortemme T
CitationJournal: Nat Nanotechnol / Year: 2013
Title: Reprogramming an ATP-driven protein machine into a light-gated nanocage.
Authors: Daniel Hoersch / Soung-Hun Roh / Wah Chiu / Tanja Kortemme /
Abstract: Natural protein assemblies have many sophisticated architectures and functions, creating nanoscale storage containers, motors and pumps. Inspired by these systems, protein monomers have been ...Natural protein assemblies have many sophisticated architectures and functions, creating nanoscale storage containers, motors and pumps. Inspired by these systems, protein monomers have been engineered to self-assemble into supramolecular architectures including symmetrical, metal-templated and cage-like structures. The complexity of protein machines, however, has made it difficult to create assemblies with both defined structures and controllable functions. Here we report protein assemblies that have been engineered to function as light-controlled nanocontainers. We show that an adenosine-5'-triphosphate-driven group II chaperonin, which resembles a barrel with a built-in lid, can be reprogrammed to open and close on illumination with different wavelengths of light. By engineering photoswitchable azobenzene-based molecules into the structure, light-triggered changes in interatomic distances in the azobenzene moiety are able to drive large-scale conformational changes of the protein assembly. The different states of the assembly can be visualized with single-particle cryo-electron microscopy, and the nanocages can be used to capture and release non-native cargos. Similar strategies that switch atomic distances with light could be used to build other controllable nanoscale machines.
History
DepositionOct 11, 2013-
Header (metadata) releaseDec 4, 2013-
Map releaseDec 4, 2013-
UpdateDec 18, 2013-
Current statusDec 18, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5769.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationchemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
Voxel sizeX=Y=Z: 1.81 Å
Density
Contour LevelBy EMDB: 0.45 / Movie #1: 0.4
Minimum - Maximum-0.18532977 - 0.67327702
Average (Standard dev.)0.00675333 (±0.07305732)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 434.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.811.811.81
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z434.400434.400434.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.1850.6730.007

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Supplemental data

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Sample components

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Entire : chemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, A...

EntireName: chemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
Components
  • Sample: chemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
  • Protein or peptide: mutant Methanococcus maripaludis chaperonin

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Supramolecule #1000: chemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, A...

SupramoleculeName: chemically cross-linked K87C/S199C Mm-cpn with ABDM (unlighted, ATP-AlFx)
type: sample / ID: 1000 / Oligomeric state: homohexadecamer / Number unique components: 1
Molecular weightExperimental: 1 MDa / Theoretical: 1 MDa / Method: size exclusion column

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Macromolecule #1: mutant Methanococcus maripaludis chaperonin

MacromoleculeName: mutant Methanococcus maripaludis chaperonin / type: protein_or_peptide / ID: 1 / Name.synonym: mutant Mm-cpn / Details: ATP-AlFx / Oligomeric state: 16-mer / Recombinant expression: Yes
Source (natural)Organism: Methanococcus maripaludis (archaea)
Molecular weightExperimental: 1 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Details: 50 mM KCl, 5 mM MgCl2, 20 mM HEPES, 10% glycerol, 0.1% OG
GridDetails: 200-mesh R1.2/1.3 holey-carbon grids, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Gatan 626 single tilt cryo transfer holder (70)
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 100 K
DateNov 28, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 173 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: particle based
Final reconstructionResolution.type: BY AUTHOR / Resolution: 17.4 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 8428
DetailsEMAN2 standard package

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