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- EMDB-3700: Structure of Rubisco from Rhodobacter sphaeroides in complex with CABP -

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Basic information

Entry
Database: EMDB / ID: EMD-3700
TitleStructure of Rubisco from Rhodobacter sphaeroides in complex with CABP
Map data
Sample
  • Complex: Rubisco
    • Protein or peptide: RbcL
    • Protein or peptide: RbcS
Biological speciesRhodobacter sphaeroides (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBracher A / Milicic G / Ciniawsky S / Wendler P / Hayer-Hartl M / Hart FU
CitationJournal: Mol Cell / Year: 2017
Title: Mechanism of Enzyme Repair by the AAA Chaperone Rubisco Activase.
Authors: Javaid Y Bhat / Goran Miličić / Gabriel Thieulin-Pardo / Andreas Bracher / Andrew Maxwell / Susanne Ciniawsky / Oliver Mueller-Cajar / John R Engen / F Ulrich Hartl / Petra Wendler / Manajit Hayer-Hartl /
Abstract: How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) ...How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco. Hydrogen/deuterium exchange and chemical crosslinking showed that Rca structurally destabilizes elements of the Rubisco active site with remarkable selectivity. Cryo-electron microscopy revealed that Rca docks onto Rubisco over one active site at a time, positioning the C-terminal strand of RbcL, which stabilizes the catalytic center, for access to the Rca hexamer pore. The pulling force of Rca is fine-tuned to avoid global destabilization and allow for precise enzyme repair.
History
DepositionMay 3, 2017-
Header (metadata) releaseMay 10, 2017-
Map releaseJul 26, 2017-
UpdateSep 20, 2017-
Current statusSep 20, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.46
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.46
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3700.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.46 / Movie #1: 0.46
Minimum - Maximum-3.4089725 - 4.2804995
Average (Standard dev.)0.0022315488 (±0.115924634)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-3.4094.2800.002

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Supplemental data

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Sample components

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Entire : Rubisco

EntireName: Rubisco
Components
  • Complex: Rubisco
    • Protein or peptide: RbcL
    • Protein or peptide: RbcS

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Supramolecule #1: Rubisco

SupramoleculeName: Rubisco / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Rubisco was treated with the inhibitor CABP
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)

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Macromolecule #1: RbcL

MacromoleculeName: RbcL / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDTKTTEIKG KERYKAGVLK YAQMGYWDGD YVPKDTDVLA LFRITPQEGV DPVEAAAAVA GESSTATWT VVWTDRLTAC DSYRAKAYRV EPVPGTPGQY FCYVAYDLIL FEEGSIANLT A SIIGNVFS FKPLKAARLE DMRFPVAYVK TYKGPPTGIV GERERLDKFG ...String:
MDTKTTEIKG KERYKAGVLK YAQMGYWDGD YVPKDTDVLA LFRITPQEGV DPVEAAAAVA GESSTATWT VVWTDRLTAC DSYRAKAYRV EPVPGTPGQY FCYVAYDLIL FEEGSIANLT A SIIGNVFS FKPLKAARLE DMRFPVAYVK TYKGPPTGIV GERERLDKFG KPLLGATTKP KL GLSGKNY GRVVYEGLKG GLDFMKDDEN INSQPFMHWR DRFLYVMEAV NLASAQTGEV KGH YLNITA GTMEEMYRRA EFAKSLGSVI VMVDLIIGYT AIQSISEWCR QNDMILHMHR AGHG TYTRQ KNHGISFRVI AKWLRLAGVD HLHCGTAVGK LEGDPLTVQG YYNVCREPFN TVDLP RGIF FEQDWADLRK VMPVASGGIH AGQMHQLLSL FGDDVVLQFG GGTIGHPMGI QAGATA NRV ALEAMVLARN EGRNIDVEGP EILRAAAKWC KPLEAALDTW GNITFNYTST DTSDFVP TA SVAM

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Macromolecule #3: RbcS

MacromoleculeName: RbcS / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MRITQGCFSF LPDLTDEQIS AQVDYCLGRG WAVSLEHTDD PHPRNTYWEM WGMPMFDLRD PKGVMIELDE CRKAWPGRYI RINAFDSTRG FETVTMSFIV NRPEVEPSLR MERTEVDGRS IRYTHSIVR

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H12ClNO3TRIS-HClTris
50.0 mMNaClSodium chlorideNaClSodium chloride
1.0 mMC10H16N5O13P3ATPAdenosine triphosphate
1.0 mMC10H12Li4N5O12P3SATP-gammaS
1.0 mMC5H12O11P2RuBP
GridModel: Quantifoil R2/2 / Material: COPPER / Support film - Material: CARBON / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsRcaCC hexamers (20 micromolar monomer) were mixed with E.C.M-CABP octamers (10 micromolar monomer) in a reaction containing 20 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MgCl2, 10 mM ATP and 1mM RuBP, for 1 min at 25oC prior to addition of 0.125 % of glutaraldehyde (GA). After 10 min the reaction was quenched by addition of 0.1M Tris HCl pH 8 followed by gel filtration on a Superdex 200 PC 3.2/30 column (GE Healthcare).The fractions were eluted in buffer A and analyzed on a 6 % native gel. Fraction 13 containing HMW complexes with the least amount of free Rubisco were chosen for cryo-EM. The crosslinked E.C.M.-CABP-RcaCC complexes were diluted to 0.0030-0.0035 g ml-1 in 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM ATP, 1 mM ATP-gammaS and 1 mM RuBP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
DetailsCs corrected Krios 1 at NeCEN (June 2016)
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Average exposure time: 1.25 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 1.3 and 1.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3 and 1.4) / Number images used: 333122

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Target criteria: Maximum likelihood

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