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- EMDB-1940: Negative stain EM density of green-type rubisco activase (R294V) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1940
TitleNegative stain EM density of green-type rubisco activase (R294V) from tobacco
Map dataNegative stain EM density of green-type rubisco activase from tobacco. Hexameric model based on p97 D2 ring is fitted into the structure.
Sample
  • Sample: Nicotiana tabacum Rubisco Activase (R294V)
  • Protein or peptide: Ribulose bisphosphate carboxylase activase 1, chloroplastic
Keywordsgreen type rubisco activase / AAA+ protein / ATPase / negative stain EM
Function / homology
Function and homology information


ribulose-1,5-bisphosphate carboxylase/oxygenase activator activity / chloroplast stroma / ATP hydrolysis activity / ATP binding / identical protein binding
Similarity search - Function
Ribulose bisphosphate carboxylase/oxygenase activase-like / : / Ribulose bisphosphate carboxylase/oxygenase activase, AAA, helical / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPase, AAA-type, core / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase/oxygenase activase 1, chloroplastic
Similarity search - Component
Biological speciesNicotiana tabacum (common tobacco)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsStotz M / Mueller-Cajar O / Ciniawsky S / Wendler P / Hartl FU / Bracher A / Hayer-Hartl M
CitationJournal: Nat Struct Mol Biol / Year: 2011
Title: Structure of green-type Rubisco activase from tobacco.
Authors: Mathias Stotz / Oliver Mueller-Cajar / Susanne Ciniawsky / Petra Wendler / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl /
Abstract: Rubisco, the enzyme that catalyzes the fixation of atmospheric CO(2) in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of ...Rubisco, the enzyme that catalyzes the fixation of atmospheric CO(2) in photosynthesis, is subject to inactivation by inhibitory sugar phosphates. Here we report the 2.95-Å crystal structure of Nicotiana tabacum Rubisco activase (Rca), the enzyme that facilitates the removal of these inhibitors. Rca from tobacco has a classical AAA(+)-protein domain architecture. Although Rca populates a range of oligomeric states when in solution, it forms a helical arrangement with six subunits per turn when in the crystal. However, negative-stain electron microscopy of the active mutant R294V suggests that Rca functions as a hexamer. The residues determining species specificity for Rubisco are located in a helical insertion of the C-terminal domain and probably function in conjunction with the N-domain in Rubisco recognition. Loop segments exposed toward the central pore of the hexamer are required for the ATP-dependent remodeling of Rubisco, resulting in the release of inhibitory sugar.
History
DepositionJul 28, 2011-
Header (metadata) releaseAug 26, 2011-
Map releaseNov 11, 2011-
UpdateDec 9, 2011-
Current statusDec 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3zw6
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

emd-1940.png

Downloads & links

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Map

FileDownload / File: emd_1940.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain EM density of green-type rubisco activase from tobacco. Hexameric model based on p97 D2 ring is fitted into the structure.
Voxel sizeX=Y=Z: 3.308 Å
Density
Contour LevelBy EMDB: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.173051 - 0.324286
Average (Standard dev.)0.000455577 (±0.00858295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 423.424 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.3083.3083.308
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z423.424423.424423.424
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-0.1730.3240.000

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Supplemental data

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Sample components

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Entire : Nicotiana tabacum Rubisco Activase (R294V)

EntireName: Nicotiana tabacum Rubisco Activase (R294V)
Components
  • Sample: Nicotiana tabacum Rubisco Activase (R294V)
  • Protein or peptide: Ribulose bisphosphate carboxylase activase 1, chloroplastic

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Supramolecule #1000: Nicotiana tabacum Rubisco Activase (R294V)

SupramoleculeName: Nicotiana tabacum Rubisco Activase (R294V) / type: sample / ID: 1000 / Oligomeric state: Hexamer / Number unique components: 1
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa / Method: DSS cross linking, gel filtration

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Macromolecule #1: Ribulose bisphosphate carboxylase activase 1, chloroplastic

MacromoleculeName: Ribulose bisphosphate carboxylase activase 1, chloroplastic
type: protein_or_peptide / ID: 1 / Name.synonym: Rubisco Activase (Rca)
Details: Hexamer assembles upon addition of 1 mM ATPgammaS at 25 deg C
Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Nicotiana tabacum (common tobacco) / synonym: Common tobacco / Organelle: Chloroplast
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pHUENtRca
SequenceInterPro: ATPase, AAA-type, core

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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

Concentration0.044 mg/mL
BufferpH: 8 / Details: 20mM Tris-HCl, 50mM NaCl, 2mM MgCl2,1mM ATPgammaS
StainingType: NEGATIVE
Details: Grids were stained twice with 2% w/v uranyl acetate
GridDetails: Plain carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.95 µm / Nominal defocus min: 0.47 µm
Sample stageSpecimen holder: Single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected for at 110k magnification
DateJun 22, 2011
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: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MRC, IMAGIC, SPIDER / Number images used: 599

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Atomic model buiding 1

Initial modelPDB ID:

3t15

SoftwareName: Chimera
DetailsProtocol: Rigid body. A module of alpha helical domain and alpha-beta domain of neighbouring subunit was overlaid with p97 D2 (3CF3) structure and the hexamer was fitted into the EM map
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Local minimisation, fit in map
Output model

PDB-3zw6:
MODEL OF HEXAMERIC AAA DOMAIN ARRANGEMENT OF GREEN-TYPE RUBISCO ACTIVASE FROM TOBACCO.

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