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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-3052 | |||||||||
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Title | Structure and mechanism of the Rubisco assembly chaperone Raf1 | |||||||||
![]() | Negative stain 3D reconstruction of crosslinked S. elongatus RbcL8 and Syn7942-Raf1 | |||||||||
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![]() | Rubisco / Chaperone / Raf1 / assembly | |||||||||
Function / homology | Chaperonin-like RbcX![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 21.4 Å | |||||||||
![]() | Hauser A / Bhat JY / Milicic G / Wendler P / Hartl FU / Bracher A / Hayer-Hartl M | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 17.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.2 KB 10.2 KB | Display Display | ![]() |
Images | ![]() | 353.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Negative stain 3D reconstruction of crosslinked S. elongatus RbcL8 and Syn7942-Raf1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.16 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Synechococcus elongatus RbcL/Raf1
Entire | Name: Synechococcus elongatus RbcL/Raf1 |
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Components |
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-Supramolecule #1000: Synechococcus elongatus RbcL/Raf1
Supramolecule | Name: Synechococcus elongatus RbcL/Raf1 / type: sample / ID: 1000 / Oligomeric state: Four Raf1 dimers bind one RbcL octamer / Number unique components: 2 |
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Molecular weight | Experimental: 740 KDa / Theoretical: 740 KDa / Method: native Mass Spectrometry |
-Macromolecule #1: Ribulose-1,5-bisphosphate carboxylase oxygenase
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Experimental: 42 KDa / Theoretical: 42 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | InterPro: Chaperonin-like RbcX |
-Macromolecule #2: Rubisco accumulation factor 1
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 40 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | negative staining |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.05 mg/mL |
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Buffer | pH: 7.5 / Details: 20mM MOPS-KOH, 100mM KCl, 5mM Mg acetate, 5mM DTT |
Staining | Type: NEGATIVE / Details: Grids were stained with 2% uranyl acetate for 30s |
Grid | Details: Plasma cleaned carbon coated grids (Quantifoil) |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
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Electron microscopy
Microscope | FEI/PHILIPS CM200FEG |
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Details | low dose |
Date | Apr 24, 2015 |
Image recording | Category: 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 |
Electron beam | Acceleration voltage: 160 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 69500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: negative stain / Specimen holder model: SIDE ENTRY, EUCENTRIC |
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Image processing
Details | particles were selected manually |
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CTF correction | Details: Relion |
Final reconstruction | Applied 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 |