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- EMDB-1655: Coupled chaperone action in folding and assembly of hexadecameric... -

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Basic information

Entry
Database: EMDB / ID: EMD-1655
TitleCoupled chaperone action in folding and assembly of hexadecameric Rubisco
Map dataThis is a cryo-EM map of the RbcL8-X8 assembly intermediate
Sample
  • Sample: Rubisco assembly intermediate containing 8 copies of the large Rubisco subunit RbcL and 8 copies of the dimeric assembly chaperone RbcX2
  • Protein or peptide: RbcL
KeywordsPhotosynthesis / protein folding / Rubisco / Rubisco assembly / chaperones
Function / homology
Function and homology information


ribulose bisphosphate carboxylase complex assembly / carboxysome / photorespiration / ribulose-bisphosphate carboxylase / carbon fixation / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / protein folding chaperone / photosynthesis / monooxygenase activity ...ribulose bisphosphate carboxylase complex assembly / carboxysome / photorespiration / ribulose-bisphosphate carboxylase / carbon fixation / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / protein folding chaperone / photosynthesis / monooxygenase activity / magnesium ion binding / protein homodimerization activity / cytoplasm
Similarity search - Function
RuBisCO chaperone RbcX / Chaperonin-like RbcX superfamily / RbcX protein / Chaperonin-like RbcX / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain / Ribulose bisphosphate carboxylase, large subunit, C-terminal ...RuBisCO chaperone RbcX / Chaperonin-like RbcX superfamily / RbcX protein / Chaperonin-like RbcX / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain / Ribulose bisphosphate carboxylase, large subunit, C-terminal / RuBisCO / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain superfamily / RuBisCO large subunit, N-terminal domain superfamily / Ribulose bisphosphate carboxylase large chain, catalytic domain
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase large chain / RuBisCO chaperone RbcX
Similarity search - Component
Biological speciesSynechococcus elongatus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsLiu C / Young A / Starling-Windhof A / Bracher A / Saschenbrecker S / Rao BV / Rao KV / Berninghausen O / Mielke T / Hartl FU ...Liu C / Young A / Starling-Windhof A / Bracher A / Saschenbrecker S / Rao BV / Rao KV / Berninghausen O / Mielke T / Hartl FU / Beckmann R / Hayer-Hartl M
CitationJournal: Nature / Year: 2010
Title: Coupled chaperone action in folding and assembly of hexadecameric Rubisco.
Authors: Cuimin Liu / Anna L Young / Amanda Starling-Windhof / Andreas Bracher / Sandra Saschenbrecker / Bharathi Vasudeva Rao / Karnam Vasudeva Rao / Otto Berninghausen / Thorsten Mielke / F Ulrich ...Authors: Cuimin Liu / Anna L Young / Amanda Starling-Windhof / Andreas Bracher / Sandra Saschenbrecker / Bharathi Vasudeva Rao / Karnam Vasudeva Rao / Otto Berninghausen / Thorsten Mielke / F Ulrich Hartl / Roland Beckmann / Manajit Hayer-Hartl /
Abstract: Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The ...Form I Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), a complex of eight large (RbcL) and eight small (RbcS) subunits, catalyses the fixation of atmospheric CO(2) in photosynthesis. The limited catalytic efficiency of Rubisco has sparked extensive efforts to re-engineer the enzyme with the goal of enhancing agricultural productivity. To facilitate such efforts we analysed the formation of cyanobacterial form I Rubisco by in vitro reconstitution and cryo-electron microscopy. We show that RbcL subunit folding by the GroEL/GroES chaperonin is tightly coupled with assembly mediated by the chaperone RbcX(2). RbcL monomers remain partially unstable and retain high affinity for GroEL until captured by RbcX(2). As revealed by the structure of a RbcL(8)-(RbcX(2))(8) assembly intermediate, RbcX(2) acts as a molecular staple in stabilizing the RbcL subunits as dimers and facilitates RbcL(8) core assembly. Finally, addition of RbcS results in RbcX(2) release and holoenzyme formation. Specific assembly chaperones may be required more generally in the formation of complex oligomeric structures when folding is closely coupled to assembly.
History
DepositionOct 20, 2009-
Header (metadata) releaseOct 27, 2009-
Map releaseJan 20, 2010-
UpdateJan 20, 2010-
Current statusJan 20, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2wvw
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_1655.jpg

Downloads & links

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Map

FileDownload / File: emd_1655.map.gz / Format: CCP4 / Size: 20.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a cryo-EM map of the RbcL8-X8 assembly intermediate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.63 Å/pix.
x 176 pix.
= 286.88 Å		1.63 Å/pix.
x 176 pix.
= 286.88 Å		1.63 Å/pix.
x 176 pix.
= 286.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.63 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.6 / Movie #1: 1.4
Minimum - Maximum-13.708500000000001 - 10.755599999999999
Average (Standard dev.)0.00000000468687 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-88-88-88
Dimensions176176176
Spacing176176176
CellA=B=C: 286.88 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.631.631.63
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z286.880286.880286.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-88-88-88
NC/NR/NS176176176
D min/max/mean-13.70910.7560.000

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Supplemental data

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Sample components

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Entire : Rubisco assembly intermediate containing 8 copies of the large Ru...

EntireName: Rubisco assembly intermediate containing 8 copies of the large Rubisco subunit RbcL and 8 copies of the dimeric assembly chaperone RbcX2
Components
  • Sample: Rubisco assembly intermediate containing 8 copies of the large Rubisco subunit RbcL and 8 copies of the dimeric assembly chaperone RbcX2
  • Protein or peptide: RbcL

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Supramolecule #1000: Rubisco assembly intermediate containing 8 copies of the large Ru...

SupramoleculeName: Rubisco assembly intermediate containing 8 copies of the large Rubisco subunit RbcL and 8 copies of the dimeric assembly chaperone RbcX2
type: sample / ID: 1000
Oligomeric state: One RbcL-octamer binds to on RbcX2 octamer
Number unique components: 1
Molecular weightTheoretical: 660 KDa

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Macromolecule #1: RbcL

MacromoleculeName: RbcL / type: protein_or_peptide / ID: 1 / Name.synonym: Rubisco large subunit / Number of copies: 8 / Oligomeric state: Octamer / Recombinant expression: Yes
Source (natural)Organism: Synechococcus elongatus (bacteria) / Strain: PCC 6301
Molecular weightTheoretical: 52.5 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pet11a

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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

BufferpH: 8.7 / Details: 20 mM Tris-HCl, pH 8.7
GridDetails: Quantifoil grids (3/3) with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 85 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: 10 sec blottting before plunging, 2 layers of filter paper

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 95 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 1.63 µm / Average electron dose: 20 e/Å2 / Details: Scanned on Heidelberg Primescan at 4000 dpi / Od range: 1.2 / Bits/pixel: 16
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38900 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder: FEI Polara cartridge system / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1 / Number images used: 11104
Final angle assignmentDetails: EMAN

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Atomic model buiding 1

Initial modelPDB ID:

3hyb

SoftwareName: manual
RefinementSpace: RECIPROCAL
Output model

PDB-2wvw:
Cryo-EM structure of the RbcL-RbcX complex

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