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- EMDB-3699: Structure of Rubisco from Rhodobacter spheroides in complex with CABP -

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Basic information

Entry
Database: EMDB / ID: EMD-3699
TitleStructure of Rubisco from Rhodobacter spheroides in complex with CABP
Map datapostprocessed Rubisco map; D4 symmetry
Sample
  • Complex: Rubisco
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain 1
  • Ligand: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordsbeta barrel / lyase
Function / homology
Function and homology information


ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / monooxygenase activity / magnesium ion binding
Similarity search - Function
Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain ...Ribulose bisphosphate carboxylase, small subunit / Ribulose bisphosphate carboxylase small subunit, domain / Ribulose bisphosphate carboxylase, small subunit superfamily / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase, small chain / Ribulose bisphosphate carboxylase large subunit, type I / Ribulose bisphosphate carboxylase, large chain, active site / Ribulose bisphosphate carboxylase large chain active site. / Ribulose bisphosphate carboxylase, large subunit, ferrodoxin-like N-terminal / Ribulose bisphosphate carboxylase large chain, N-terminal domain / Ribulose bisphosphate carboxylase, large subunit, C-terminal / RuBisCO / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain superfamily / RuBisCO large subunit, N-terminal domain superfamily / Ribulose bisphosphate carboxylase large chain, catalytic domain
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase large chain / Ribulose bisphosphate carboxylase small subunit
Similarity search - Component
Biological speciesRhodobacter sphaeroides (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsBracher A / Milicic G
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-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5nv3
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3699.png

Downloads & links

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Map

FileDownload / File: emd_3699.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpostprocessed Rubisco map; D4 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å		1.04 Å/pix.
x 320 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.04 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9 / Movie #1: 0.9
Minimum - Maximum-8.962299 - 10.2346325
Average (Standard dev.)0.0022220209 (±0.27295503)
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-8.96210.2350.002

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Supplemental data

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Additional map: unfiltered Rubisco map; D4 symmetry

Fileemd_3699_additional.map
Annotationunfiltered Rubisco map; D4 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rubisco

EntireName: Rubisco
Components
  • Complex: Rubisco
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain 1
  • Ligand: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Rubisco

SupramoleculeName: Rubisco / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Rubisco was treated with the inhibitor CABP.
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)

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Macromolecule #1: Ribulose bisphosphate carboxylase large chain

MacromoleculeName: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Molecular weightTheoretical: 51.768852 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: RYKAGVLKYA QMGYWDGDYV PKDTDVLALF RITPQEGVDP VEAAAAVAGE SSTATWTVVW TDRLTACDSY RAKAYRVEPV PGTPGQYFC YVAYDLILFE EGSIANLTAS IIGNVFSFKP LKAARLEDMR FPVAYVKTYK GPPTGIVGER ERLDKFGKPL L GATTKPKL ...String:
RYKAGVLKYA QMGYWDGDYV PKDTDVLALF RITPQEGVDP VEAAAAVAGE SSTATWTVVW TDRLTACDSY RAKAYRVEPV PGTPGQYFC YVAYDLILFE EGSIANLTAS IIGNVFSFKP LKAARLEDMR FPVAYVKTYK GPPTGIVGER ERLDKFGKPL L GATTKPKL GLSGKNYGRV VYEGLKGGLD FM(KCX)DDENINS QPFMHWRDRF LYVMEAVNLA SAQTGEVKGH YLNITAGT M EEMYRRAEFA KSLGSVIVMV DLIIGYTAIQ SISEWCRQND MILHMHRAGH GTYTRQKNHG ISFRVIAKWL RLAGVDHLH CGTAVGKLEG DPLTVQGYYN VCREPFNTVD LPRGIFFEQD WADLRKVMPV ASGGIHAGQM HQLLSLFGDD VVLQFGGGTI GHPMGIQAG ATANRVALEA MVLARNEGRN IDVEGPEILR AAAKWCKPLE AALDTWGNIT FNYTSTDTSD FV

UniProtKB: Ribulose bisphosphate carboxylase large chain

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Macromolecule #2: Ribulose bisphosphate carboxylase small chain 1

MacromoleculeName: Ribulose bisphosphate carboxylase small chain 1 / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Molecular weightTheoretical: 15.183234 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MRITQGCFSF LPDLTDEQIS AQVDYCLGRG WAVSLEHTDD PHPRNTYWEM WGMPMFDLRD PKGVMIELDE CRKAWPGRYI RINAFDSTR GFETVTMSFI VNRPEVEPSL RMERTEVDGR SIRYTHSIVR

UniProtKB: Ribulose bisphosphate carboxylase small subunit

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Macromolecule #3: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

MacromoleculeName: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 8 / Formula: CAP
Molecular weightTheoretical: 356.115 Da
Chemical component information

ChemComp-CAP:
2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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-HCl
50.0 mMNaClNaCl
1.0 mMC10H16N5O13P3ATP
1.0 mMC10H12Li4N5O12P3SATP-gammaS
1.0 mMC5H12O11P2RuBP
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
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1bxn

Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 333122
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER / Software - Name: RELION

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Atomic model buiding 1

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

PDB-5nv3:
Structure of Rubisco from Rhodobacter sphaeroides in complex with CABP

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