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- EMDB-1531: Three-dimensional structure of Aquifex aeolicus co-chaperonin pro... -

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Basic information

Entry
Database: EMDB / ID: EMD-1531
TitleThree-dimensional structure of Aquifex aeolicus co-chaperonin protein 10 complexed with GroEL and ADP by cryo-EM
Map datanone
Sample
  • Sample: Aacpn10 capped to one end of GroEL under Mg-ADP
  • Protein or peptide: Chaperonin
  • Protein or peptide: Aquifex aeolicus co-chaperonin 10
Keywordsco-chaperonin / hyper-thermophile / conformational heterogeneity / electron cryomicroscopy
Function / homology:
Function and homology information
Biological speciesEscherichia coli (E. coli) / Aquifex aeolicus (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.0 Å
AuthorsChen DH / Luke K / Zhang J / Chiu W / Wittung-Stafshede P
CitationJournal: J Mol Biol / Year: 2008
Title: Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques.
Authors: Dong-Hua Chen / Kathryn Luke / Junjie Zhang / Wah Chiu / Pernilla Wittung-Stafshede /
Abstract: Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the ...Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-A resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.
History
DepositionJun 25, 2008-
Header (metadata) releaseJun 25, 2008-
Map releaseJun 11, 2009-
UpdateJan 22, 2014-
Current statusJan 22, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.93
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.93
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1aon
  • Surface level: 0.93
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1531.gif

Downloads & links

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Map

FileDownload / File: emd_1531.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 200 pix.
= 359. Å		1.8 Å/pix.
x 200 pix.
= 359. Å		1.8 Å/pix.
x 200 pix.
= 359. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.795 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.0 / Movie #1: 0.93
Minimum - Maximum-0.87262 - 1.67616
Average (Standard dev.)0.0299941 (±0.161115)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 359 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.7951.7951.795
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z359.000359.000359.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-75-75-74
NX/NY/NZ150150150
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.8731.6760.030

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Supplemental data

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Sample components

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Entire : Aacpn10 capped to one end of GroEL under Mg-ADP

EntireName: Aacpn10 capped to one end of GroEL under Mg-ADP
Components
  • Sample: Aacpn10 capped to one end of GroEL under Mg-ADP
  • Protein or peptide: Chaperonin
  • Protein or peptide: Aquifex aeolicus co-chaperonin 10

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Supramolecule #1000: Aacpn10 capped to one end of GroEL under Mg-ADP

SupramoleculeName: Aacpn10 capped to one end of GroEL under Mg-ADP / type: sample / ID: 1000
Details: Aacpn10 was incubated with GroEL in 50 mM Tris-HCl, 30 mM MgCl2, 2 mM ADP, pH 7.5 at 37 Celsius for 1 hour
Oligomeric state: One heptamer of Aacpn10 and seven ADP bind to one heptamer of GroEL
Number unique components: 2
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa

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Macromolecule #1: Chaperonin

MacromoleculeName: Chaperonin / type: protein_or_peptide / ID: 1 / Name.synonym: GroEL / Number of copies: 1 / Oligomeric state: heptamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pMESL
SequenceInterPro: INTERPRO: IPR012723

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Macromolecule #2: Aquifex aeolicus co-chaperonin 10

MacromoleculeName: Aquifex aeolicus co-chaperonin 10 / type: protein_or_peptide / ID: 2 / Name.synonym: Aacpn10 / Number of copies: 1 / Oligomeric state: heptamer / Recombinant expression: Yes
Source (natural)Organism: Aquifex aeolicus (bacteria) / Strain: AQ2199 / Tissue: Bacterial / Cell: Aquifex aeolicus / Location in cell: Cytosol
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET24c

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 7.5 / Details: 50 mM Tris-HCl, 30 mM MgCl2
StainingType: NEGATIVE
Details: The sample solution was blotted for 2.5 s before submersion in liquid ethane cooled by liquid nitrogen using FEI Vitrobot
GridDetails: 400 mesh copper grid (R1.2/1.3 Quantifoil)
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 4.2 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: blot for 2.5 s before plunging

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Electron microscopy

MicroscopeJEOL 3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 3.4 µm / Nominal defocus min: 0.86 µm / Nominal magnification: 50000
Sample stageSpecimen holder: top-entry / Specimen holder model: OTHER
TemperatureMin: 4.2 K / Max: 4.2 K / Average: 4.2 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 400,000 times magnification
DetailsYoshi MDS box for low-dose imaging
DateFeb 16, 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: 720 / Average electron dose: 36 e/Å2
Details: The particle images were averaged by 2 to give 1.91 Angstrom per pixel. The final corrected pixel size for the 3D density map is 1.8 Angstrom per pixel.
Bits/pixel: 8

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Image processing

CTF correctionDetails: each micrograph
Final two d classificationNumber classes: 514
Final angle assignmentDetails: EMAN
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN
Details: The final map was calculated from the class averages and was filtered to show the subnanometer resolution features.
Number images used: 10772
DetailsThe ratio of GroEL monomers to Aacpn10 monomers was about 1

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Atomic model buiding 1

Initial modelPDB ID:

1aon


Chain - Chain ID: A
SoftwareName: UROX
DetailsPDBEntryID_givenInChain. Protocol: domain-based rigid body. UROX was used to perform the domain-as-rigid-body fitting of the GroEL-GroES-ADP crystal structure into the cryo-EM density map of the GroEL-Aacpn10-ADP complex
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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