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- EMDB-1932: Negative stain EM Map of the AAA protein CbbX, a red-type Rubisco... -

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Basic information

Entry
Database: EMDB / ID: EMD-1932
TitleNegative stain EM Map of the AAA protein CbbX, a red-type Rubisco activase from R. sphaeroides
Map dataNegative stain EM reconstruction of the R. sphaeroides CbbX hexamer
Sample
  • Sample: R. sphaeroides CbbX 3D density map.
  • Protein or peptide: Rubisco ActivaseRuBisCO
KeywordsAAA+ protein / ATPase / Rubisco activase
Function / homology
Function and homology information


ATP hydrolysis activity / ATP binding
Similarity search - Function
CbxX/CfxQ, monofunctional / CbxX/CfxQ / CbbX, AAA lid domain / AAA lid domain / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesRhodobacter sphaeroides (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsMueller-Cajar O / Stotz M / Wendler P / Hartl FU / Bracher A / Hayer-Hartl M
CitationJournal: Nature / Year: 2011
Title: Structure and function of the AAA+ protein CbbX, a red-type Rubisco activase.
Authors: Oliver Mueller-Cajar / Mathias Stotz / Petra Wendler / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl /
Abstract: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5- ...Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the fixation of atmospheric CO(2) in photosynthesis, but tends to form inactive complexes with its substrate ribulose 1,5-bisphosphate (RuBP). In plants, Rubisco is reactivated by the AAA(+) (ATPases associated with various cellular activities) protein Rubisco activase (Rca), but no such protein is known for the Rubisco of red algae. Here we identify the protein CbbX as an activase of red-type Rubisco. The 3.0-Å crystal structure of unassembled CbbX from Rhodobacter sphaeroides revealed an AAA(+) protein architecture. Electron microscopy and biochemical analysis showed that ATP and RuBP must bind to convert CbbX into functionally active, hexameric rings. The CbbX ATPase is strongly stimulated by RuBP and Rubisco. Mutational analysis suggests that CbbX functions by transiently pulling the carboxy-terminal peptide of the Rubisco large subunit into the hexamer pore, resulting in the release of the inhibitory RuBP. Understanding Rubisco activation may facilitate efforts to improve CO(2) uptake and biomass production by photosynthetic organisms.
History
DepositionJul 18, 2011-
Header (metadata) releaseAug 26, 2011-
Map releaseNov 3, 2011-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3zuh
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-1932.png

emd_1932.png

Downloads & links

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Map

FileDownload / File: emd_1932.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM reconstruction of the R. sphaeroides CbbX hexamer
Voxel sizeX=Y=Z: 3.31 Å
Density
Contour LevelBy EMDB: 0.005 / Movie #1: 0.017
Minimum - Maximum-0.011328 - 0.197363
Average (Standard dev.)0.000343572 (±0.00533179)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 423.68 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.313.313.31
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z423.680423.680423.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0110.1970.000

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Supplemental data

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Sample components

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Entire : R. sphaeroides CbbX 3D density map.

EntireName: R. sphaeroides CbbX 3D density map.
Components
  • Sample: R. sphaeroides CbbX 3D density map.
  • Protein or peptide: Rubisco ActivaseRuBisCO

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Supramolecule #1000: R. sphaeroides CbbX 3D density map.

SupramoleculeName: R. sphaeroides CbbX 3D density map. / type: sample / ID: 1000 / Oligomeric state: Hexamer / Number unique components: 1
Molecular weightTheoretical: 206 KDa

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Macromolecule #1: Rubisco Activase

MacromoleculeName: Rubisco Activase / type: protein_or_peptide / ID: 1 / Name.synonym: CbbX
Details: The protein is bound to Ribulose-1,5-bisphosphate, ATP and ATPgammaS
Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Molecular weightTheoretical: 206 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pHue
SequenceGO: ATP binding

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.072 mg/mL
BufferpH: 8
Details: 20 mM Tris pH 8.0, 50 mM NaCl, 5mM MgCl2, 1mM Ribulose-1,5-bisphosphate, 1mM ATP/ATPgammaS
StainingType: NEGATIVE
Details: Grids with adsorbed protein stained with 2% (w/v) uranyl acetate for 40 seconds.
GridDetails: plain carbon grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 90600 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.26 µm / Nominal magnification: 90600
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected for at 110k magnification
DateNov 19, 2010
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (2k x 2k) / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: phase flipping, each particle
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MRC, IMAGIC, SPIDER / Number images used: 245

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Atomic model buiding 1

Initial modelPDB ID:

3syl

DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3zuh:
Negative stain EM Map of the AAA protein CbbX, a red-type Rubisco activase from R. sphaeroides

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Atomic model buiding 2

Initial modelPDB ID:

3cf3

DetailsProtocol: rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3zuh:
Negative stain EM Map of the AAA protein CbbX, a red-type Rubisco activase from R. sphaeroides

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