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- EMDB-2325: Visualizing GroEL/ES in the Act of Encapsulating a Non-Native Sub... -

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Entry
Database: EMDB / ID: EMD-2325
TitleVisualizing GroEL/ES in the Act of Encapsulating a Non-Native Substrate Protein
Map dataC7-symmetry imposed 3D reconstruction of the bullet-shaped subpopulation sorted from the heterogeneous sample of EL43Py reacted with GroES and nucleotide ATP
Sample
  • Sample: GroEL variant EL43Py capped by GroES with the assistance of nucleotide ATP
  • Protein or peptide: GroELcys0-D398A-S43C-pyrene
  • Protein or peptide: GroES
  • Ligand: Adenosine triphosphate
Keywordsprotein folding / chaperonin / GroEL / GroEL-GroES / cryo-EM / heterogeneity
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) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.9 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: 1.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3zpz
  • Surface level: 1.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2325.png

Downloads & links

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Map

FileDownload / File: emd_2325.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC7-symmetry imposed 3D reconstruction of the bullet-shaped subpopulation sorted from the heterogeneous sample of EL43Py reacted with GroES and nucleotide ATP
Voxel sizeX=Y=Z: 2.08 Å
Density
Contour LevelBy AUTHOR: 1.3 / Movie #1: 1.3
Minimum - Maximum-1.13611758 - 3.1620748
Average (Standard dev.)0.03553469 (±0.23130623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.082.082.08
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z374.400374.400374.400
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-1.1363.1620.036

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Supplemental data

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Sample components

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Entire : GroEL variant EL43Py capped by GroES with the assistance of nucle...

EntireName: GroEL variant EL43Py capped by GroES with the assistance of nucleotide ATP
Components
  • Sample: GroEL variant EL43Py capped by GroES with the assistance of nucleotide ATP
  • Protein or peptide: GroELcys0-D398A-S43C-pyrene
  • Protein or peptide: GroES
  • Ligand: Adenosine triphosphate

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Supramolecule #1000: GroEL variant EL43Py capped by GroES with the assistance of nucle...

SupramoleculeName: GroEL variant EL43Py capped by GroES with the assistance of nucleotide ATP
type: sample / ID: 1000 / Details: The molecule is bullet-shaped
Oligomeric state: One heptamer of GroES and seven nucleotides of ATP binds to one tetradecamer of GroEL variant EL43Py
Number unique components: 3
Molecular weightExperimental: 870 KDa / Theoretical: 870 KDa
Method: GroEL molecular weight 800kDa plus GroES molecular weight 70kDa

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Macromolecule #1: GroELcys0-D398A-S43C-pyrene

MacromoleculeName: GroELcys0-D398A-S43C-pyrene / type: protein_or_peptide / ID: 1 / Name.synonym: EL398A43Py
Details: A GroEL variant in which the endogenous Cys residues (138, 458, 519) have been changed to Ala (GroELcys0) was modified to contain two additional mutations: D398A and S43C. The D398A mutation ...Details: A GroEL variant in which the endogenous Cys residues (138, 458, 519) have been changed to Ala (GroELcys0) was modified to contain two additional mutations: D398A and S43C. The D398A mutation prevents ATP hydrolysis by GroEL, while the S43C mutation, located at the tip of stem loop at the bottom of the GroEL cavity, permits the covalent attachment of N-1-pyrene maleimide to this position.
Number of copies: 14 / Oligomeric state: Tetradecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: Cytoplasm
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (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 (bacteria) / Recombinant plasmid: pACYC
SequenceUniProtKB: Co-chaperonin GroES

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Macromolecule #3: Adenosine triphosphate

MacromoleculeName: Adenosine triphosphate / type: ligand / ID: 3 / Name.synonym: ATP / Number of copies: 7 / 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

Concentration6.4 mg/mL
BufferpH: 7.6 / Details: 50 mM Hepes, 5 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 3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 61060 / Illumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 1.6 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Helium cooled top-entry cartridge / Specimen holder model: JEOL
TemperatureMin: 10 K / Max: 12 K / Average: 11 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 400,000 times magnification
DateMar 7, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 390 / Average electron dose: 36 e/Å2 / Details: The scanned images were averaged twice. / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each micrograph
Final two d classificationNumber classes: 257
Final angle assignmentDetails: EMAN1
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: OTHER / Software - Name: EMAN1 / Number images used: 8372
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 of greatest interest.

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

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