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- EMDB-6477: Identification and characterization of multiple Rubisco activases... -

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Basic information

Entry
Database: EMDB / ID: EMD-6477
TitleIdentification and characterization of multiple Rubisco activases in chemoautotrophic bacteria
Map dataReconstruction of AfQ2
Sample
  • Sample: AfQ2
  • Protein or peptide: RuBisCo activaseRuBisCO
KeywordsRubisco / activase
Biological speciesAcidithiobacillus ferrooxidans (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsTsai Y-CC / Lapina MC / Mueller-Cajar O / Bhushan S
CitationJournal: Nat Commun / Year: 2015
Title: Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria.
Authors: Yi-Chin Candace Tsai / Maria Claribel Lapina / Shashi Bhushan / Oliver Mueller-Cajar /
Abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) ...Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation.
History
DepositionOct 5, 2015-
Header (metadata) releaseNov 11, 2015-
Map releaseNov 18, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.86
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.86
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6477.gif

Downloads & links

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Map

FileDownload / File: emd_6477.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of AfQ2
Voxel sizeX=Y=Z: 2.11 Å
Density
Contour LevelBy AUTHOR: 2.86 / Movie #1: 2.86
Minimum - Maximum-4.41357613 - 8.501306530000001
Average (Standard dev.)0.15246363 (±0.92950153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 210.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.112.112.11
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z211.000211.000211.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-4.4148.5010.152

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Supplemental data

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Sample components

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Entire : AfQ2

EntireName: AfQ2
Components
  • Sample: AfQ2
  • Protein or peptide: RuBisCo activaseRuBisCO

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Supramolecule #1000: AfQ2

SupramoleculeName: AfQ2 / type: sample / ID: 1000 / Oligomeric state: Hexamer / Number unique components: 1
Molecular weightExperimental: 173 KDa / Theoretical: 181 KDa / Method: sedimentation and gel filtration

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Macromolecule #1: RuBisCo activase

MacromoleculeName: RuBisCo activase / type: protein_or_peptide / ID: 1 / Name.synonym: CbbQ / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Acidithiobacillus ferrooxidans (bacteria) / synonym: Chemoautotrophic bacteria / Location in cell: Cytosol
Molecular weightExperimental: 173 KDa / Theoretical: 181 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 / Recombinant plasmid: pHue

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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

Concentration0.05 mg/mL
BufferpH: 8 / Details: 50 mM NaCl, 20 mM Tris-HCl, 5 mM Mg-ATP
StainingType: NEGATIVE
Details: Negative stain, 2% w/v uranyl acetate for 30 seconds
GridDetails: 200 mesh Cu grid with thin carbon support, glow discharged
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: 66350 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 6.3 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 43000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 66,000 times magnification.
DateApr 29, 2014
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 110 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: Each particle
Final two d classificationNumber classes: 20
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: EMAN2 / Number images used: 958
DetailsEMAN2

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