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- EMDB-2327: Visualizing GroEL/ES in the Act of Encapsulating a Non-Native Sub... -

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Entry
Database: EMDB / ID: EMD-2327
TitleVisualizing GroEL/ES in the Act of Encapsulating a Non-Native Substrate Protein
Map dataSymmetry-free 3D reconstruction of the bullet-shaped substrate-encapsulated subpopulation sorted from the heterogeneous sample of wild-type GroEL reacted with the substrate protein RuBisCO, GroES and nucleotide ATP
Sample
  • Sample: Non-native RuBisCO substrate protein encapsulated inside the cavity of GroEL capped by GroES with the assistance of nucleotide ATP
  • Protein or peptide: GroEL-D398A
  • Protein or peptide: GroES
  • Protein or peptide: Ribulose-1,5-bisphosphate carboxylase oxygenase
  • Ligand: Adenosine triphosphate
Keywordsprotein folding / chaperonin / GroEL / GroEL-GroES / cryo-EM / heterogeneity / substrate / RuBisCO / encapsulation
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / protein folding chaperone / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein-folding chaperone binding / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / metal ion binding / cytosol
Similarity search - Function
Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily ...Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / GroES-like superfamily
Similarity search - Domain/homology
Chaperonin GroEL / Co-chaperonin GroES
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Rhodospirillum rubrum (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.9 Å
AuthorsChen D-H / Madan D / Weaver J / Lin Z / Schroder GF / Chiu W / Rye HS
CitationJournal: Cell / Year: 2013
Title: Visualizing GroEL/ES in the act of encapsulating a folding protein.
Authors: Dong-Hua Chen / Damian Madan / Jeremy Weaver / Zong Lin / Gunnar F Schröder / Wah Chiu / Hays S Rye /
Abstract: The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry- ...The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.
History
DepositionMar 1, 2013-
Header (metadata) releaseMar 13, 2013-
Map releaseMar 13, 2013-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3zq1
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2327.png

Downloads & links

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Map

FileDownload / File: emd_2327.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetry-free 3D reconstruction of the bullet-shaped substrate-encapsulated subpopulation sorted from the heterogeneous sample of wild-type GroEL reacted with the substrate protein RuBisCO, GroES and nucleotide ATP
Voxel sizeX=Y=Z: 2.12 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.07791637 - 1.79304695
Average (Standard dev.)0.04864103 (±0.21416679)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 381.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z381.600381.600381.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-12-40
NX/NY/NZ732581
MAP C/R/S123
start NC/NR/NS-90-90-90
NC/NR/NS180180180
D min/max/mean-0.0781.7930.049

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Supplemental data

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Sample components

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Entire : Non-native RuBisCO substrate protein encapsulated inside the cavi...

EntireName: Non-native RuBisCO substrate protein encapsulated inside the cavity of GroEL capped by GroES with the assistance of nucleotide ATP
Components
  • Sample: Non-native RuBisCO substrate protein encapsulated inside the cavity of GroEL capped by GroES with the assistance of nucleotide ATP
  • Protein or peptide: GroEL-D398A
  • Protein or peptide: GroES
  • Protein or peptide: Ribulose-1,5-bisphosphate carboxylase oxygenase
  • Ligand: Adenosine triphosphate

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Supramolecule #1000: Non-native RuBisCO substrate protein encapsulated inside the cavi...

SupramoleculeName: Non-native RuBisCO substrate protein encapsulated inside the cavity of GroEL capped by GroES with the assistance of nucleotide ATP
type: sample / ID: 1000 / Details: The molecule is bullet-shaped
Oligomeric state: Non-native RuBisCO substrate protein was encapsulated inside the cavity formed by one tetradecamer of GroEL, one heptamer of GroES and seven nucleotides of ATP
Number unique components: 4
Molecular weightExperimental: 920 KDa / Theoretical: 920 KDa
Method: Estimated by the sum of GroEL molecular weight 800kDa, GroES molecular weight 70kDa and one RuBisCO monomer molecular weight 50kDa

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Macromolecule #1: GroEL-D398A

MacromoleculeName: GroEL-D398A / type: protein_or_peptide / ID: 1 / Name.synonym: EL398A
Details: The D398A mutation prevents ATP hydrolysis by GroEL.
Number of copies: 14 / Oligomeric state: Tetradecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: BL21 / Location in cell: Cytoplasm
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pACYC
SequenceUniProtKB: Chaperonin GroEL

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Macromolecule #2: GroES

MacromoleculeName: GroES / type: protein_or_peptide / ID: 2 / Number of copies: 7 / Oligomeric state: Heptamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: Cytoplasm
Molecular weightExperimental: 70 KDa / Theoretical: 70 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pACYC
SequenceUniProtKB: Co-chaperonin GroES

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Macromolecule #3: Ribulose-1,5-bisphosphate carboxylase oxygenase

MacromoleculeName: Ribulose-1,5-bisphosphate carboxylase oxygenase / type: protein_or_peptide / ID: 3 / Name.synonym: RuBisCO
Details: The native RuBisCO is a dimer, but the encapsulated RuBisCO is a non-native monomer.
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Rhodospirillum rubrum (bacteria) / Location in cell: cytoplasm
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pUC derived plasmid with T7 promoter

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Macromolecule #4: Adenosine triphosphate

MacromoleculeName: Adenosine triphosphate / type: ligand / ID: 4 / Name.synonym: ATP / Recombinant expression: No
Source (natural)Organism: synthetic construct (others)

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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

Concentration2.4 mg/mL
BufferpH: 7.6 / Details: 50 mM Hepes, 50 mM KOAc, 10 mM Mg(OAc)2, 2 mM DTT
GridDetails: 400 mesh R1.2/1.3 Quantifoil grid glow-discharged 10 sec before freezing
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 98 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 1 second before plunging

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 70760 / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: JEOL / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
TemperatureMin: 100.9 K / Max: 101.1 K / Average: 101 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateDec 21, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Digitization - Sampling interval: 15 µm / Number real images: 1537 / Average electron dose: 20 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each frame
Final two d classificationNumber classes: 280
Final angle assignmentDetails: EMAN1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.9 Å / Resolution method: OTHER / Software - Name: EMAN1 / Number images used: 8189
DetailsParticle selection was performed semi-automatically using the EMAN1 program, boxer. The contrast transfer function fitting was performed automatically using the program fitctf.py and then fine-tuned manually using the EMAN1 program ctfit. The methodology of EMAN1 multiple-model refinement (EMAN1 program multirefine) for compositionally and conformationally heterogeneous complex analysis was used to sort out the relatively homogeneous bullet-shaped particle images with the substrate protein inside the cis cavity.

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Atomic model buiding 1

Initial modelPDB ID:

1aon


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L / Chain - #12 - Chain ID: M / Chain - #13 - Chain ID: N / Chain - #14 - Chain ID: O / Chain - #15 - Chain ID: P / Chain - #16 - Chain ID: Q / Chain - #17 - Chain ID: R / Chain - #18 - Chain ID: S / Chain - #19 - Chain ID: T / Chain - #20 - Chain ID: U
SoftwareName: DireX
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation coefficient
Output model

PDB-3zq1:
Visualizing GroEL-ES in the Act of Encapsulating a Non-Native Substrate Protein

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