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- EMDB-3052: Structure and mechanism of the Rubisco assembly chaperone Raf1 -

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Basic information

Entry
Database: EMDB / ID: EMD-3052
TitleStructure and mechanism of the Rubisco assembly chaperone Raf1
Map dataNegative stain 3D reconstruction of crosslinked S. elongatus RbcL8 and Syn7942-Raf1
Sample
  • Sample: Synechococcus elongatus RbcL/Raf1
  • Protein or peptide: Ribulose-1,5-bisphosphate carboxylase oxygenase
  • Protein or peptide: Rubisco accumulation factor 1
KeywordsRubisco / Chaperone / Raf1 / assembly
Function / homologyChaperonin-like RbcX
Function and homology information
Biological speciesSynechococcus elongatus PCC 6301 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 21.4 Å
AuthorsHauser A / Bhat JY / Milicic G / Wendler P / Hartl FU / Bracher A / Hayer-Hartl M
CitationJournal: Nat Struct Mol Biol / Year: 2015
Title: Structure and mechanism of the Rubisco-assembly chaperone Raf1.
Authors: Thomas Hauser / Javaid Y Bhat / Goran Miličić / Petra Wendler / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl /
Abstract: Biogenesis of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits, requires assembly chaperones. Here we analyzed the role of Rubisco accumulation ...Biogenesis of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits, requires assembly chaperones. Here we analyzed the role of Rubisco accumulation factor1 (Raf1), a dimer of ∼40-kDa subunits. We find that Raf1 from Synechococcus elongatus acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by RbcS results in holoenzyme formation. Crystal structures show that Raf1 from Arabidopsis thaliana consists of a β-sheet dimerization domain and a flexibly linked α-helical domain. Chemical cross-linking and EM reconstruction indicate that the β-domains bind along the equator of each RbcL2 unit, and the α-helical domains embrace the top and bottom edges of RbcL2. Raf1 fulfills a role similar to that of the assembly chaperone RbcX, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.
History
DepositionJun 19, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseAug 5, 2015-
UpdateSep 23, 2015-
Current statusSep 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.033
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.033
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMDB3052.png

Downloads & links

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Map

FileDownload / File: emd_3052.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain 3D reconstruction of crosslinked S. elongatus RbcL8 and Syn7942-Raf1
Voxel sizeX=Y=Z: 2.16 Å
Density
Contour LevelBy EMDB: 0.035 / Movie #1: 0.033
Minimum - Maximum-0.05337655 - 0.07752982
Average (Standard dev.)-0.00118651 (±0.00975322)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z414.720414.720414.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-21-120
NX/NY/NZ432573
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0530.078-0.001

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Supplemental data

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Sample components

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Entire : Synechococcus elongatus RbcL/Raf1

EntireName: Synechococcus elongatus RbcL/Raf1
Components
  • Sample: Synechococcus elongatus RbcL/Raf1
  • Protein or peptide: Ribulose-1,5-bisphosphate carboxylase oxygenase
  • Protein or peptide: Rubisco accumulation factor 1

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Supramolecule #1000: Synechococcus elongatus RbcL/Raf1

SupramoleculeName: Synechococcus elongatus RbcL/Raf1 / type: sample / ID: 1000 / Oligomeric state: Four Raf1 dimers bind one RbcL octamer / Number unique components: 2
Molecular weightExperimental: 740 KDa / Theoretical: 740 KDa / Method: native Mass Spectrometry

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Macromolecule #1: Ribulose-1,5-bisphosphate carboxylase oxygenase

MacromoleculeName: Ribulose-1,5-bisphosphate carboxylase oxygenase / type: protein_or_peptide / ID: 1 / Name.synonym: RuBisCO
Details: complex is cross linked with bifunctional crosslinker disuccinimidylsuberate (DSS)
Number of copies: 8 / Oligomeric state: Octamer / Recombinant expression: Yes
Source (natural)Organism: Synechococcus elongatus PCC 6301 (bacteria) / Organelle: chloroplast
Molecular weightExperimental: 42 KDa / Theoretical: 42 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceInterPro: Chaperonin-like RbcX

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Macromolecule #2: Rubisco accumulation factor 1

MacromoleculeName: Rubisco accumulation factor 1 / type: protein_or_peptide / ID: 2 / Name.synonym: Raf1
Details: complex is cross linked with bifunctional crosslinker disuccinimidylsuberate (DSS)
Number of copies: 8 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Synechococcus elongatus PCC 6301 (bacteria) / Organelle: Chloroplast
Molecular weightTheoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / 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: 7.5 / Details: 20mM MOPS-KOH, 100mM KCl, 5mM Mg acetate, 5mM DTT
StainingType: NEGATIVE / Details: Grids were stained with 2% uranyl acetate for 30s
GridDetails: Plasma cleaned carbon coated grids (Quantifoil)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 69500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 50000
Sample stageSpecimen holder: negative stain / Specimen holder model: SIDE ENTRY, EUCENTRIC
Detailslow dose
DateApr 24, 2015
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 49 / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: Relion
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 21.4 Å / Resolution method: OTHER / Software - Name: MRC, RELION, 1.3 / Number images used: 5183
Detailsparticles were selected manually

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