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

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Basic information

Entry
Database: PDB / ID: 5nv3
TitleStructure of Rubisco from Rhodobacter sphaeroides in complex with CABP
Components
  • Ribulose bisphosphate carboxylase large chain
  • Ribulose bisphosphate carboxylase small chain 1
KeywordsLYASE / beta barrel
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 1,5 Bisphosphate Carboxylase/Oxygenase / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain / RuBisCO large subunit, 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, small subunit / Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase / Ribulose bisphosphate carboxylase, large subunit, C-terminal domain / RuBisCO large subunit, 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 / Alpha-Beta Plaits / TIM Barrel / Alpha-Beta Barrel / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / Ribulose bisphosphate carboxylase large chain / Ribulose bisphosphate carboxylase small subunit
Similarity search - Component
Biological speciesRhodobacter sphaeroides (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsBracher, A. / Milicic, G. / Ciniawsky, S. / Wendler, P. / Hayer-Hartl, M. / Hartl, F.U.
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, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 26, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Database references / Refinement description / Category: citation / em_3d_fitting
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _em_3d_fitting.target_criteria
Revision 1.2Sep 20, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Ribulose bisphosphate carboxylase large chain
I: Ribulose bisphosphate carboxylase small chain 1
B: Ribulose bisphosphate carboxylase large chain
J: Ribulose bisphosphate carboxylase small chain 1
C: Ribulose bisphosphate carboxylase large chain
K: Ribulose bisphosphate carboxylase small chain 1
D: Ribulose bisphosphate carboxylase large chain
L: Ribulose bisphosphate carboxylase small chain 1
E: Ribulose bisphosphate carboxylase large chain
M: Ribulose bisphosphate carboxylase small chain 1
F: Ribulose bisphosphate carboxylase large chain
N: Ribulose bisphosphate carboxylase small chain 1
G: Ribulose bisphosphate carboxylase large chain
O: Ribulose bisphosphate carboxylase small chain 1
H: Ribulose bisphosphate carboxylase large chain
P: Ribulose bisphosphate carboxylase small chain 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)538,66032
Polymers535,61716
Non-polymers3,04316
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area109360 Å2
ΔGint-474 kcal/mol
Surface area126350 Å2
MethodPISA

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Components

#1: Protein
Ribulose bisphosphate carboxylase large chain / RuBisCO large subunit


Mass: 51768.852 Da / Num. of mol.: 8 / Fragment: RbcL
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodobacter sphaeroides (bacteria) / Gene: cbbL, cbbL1, rbcL / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P27997, ribulose-bisphosphate carboxylase
#2: Protein
Ribulose bisphosphate carboxylase small chain 1 / RuBisCO small subunit 1


Mass: 15183.234 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodobacter sphaeroides (bacteria) / Gene: cbbS, rbcS / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P27998, ribulose-bisphosphate carboxylase
#3: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharide / Mass: 356.115 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C6H14O13P2 / References: ribulose-bisphosphate carboxylase
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rubisco / Type: COMPLEX / Details: Rubisco was treated with the inhibitor CABP. / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rhodobacter sphaeroides (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRiS-HClC4H12ClNO31
250 mMNaClNaCl1
31 mMATPC10H16N5O13P31
41 mMATP-gammaSC10H12Li4N5O12P3S1
51 mMRuBPC5H12O11P21
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: RcaCC 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 ...Details: RcaCC 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
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs corrected Krios 1 at NeCEN (June 2016)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingAverage exposure time: 1.25 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Image scansMovie frames/image: 22

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Processing

EM software
IDNameVersionCategory
4CTFFIND4CTF correction
7Coot0.8.2model fitting
9REFMAC5.8.0155model refinement
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 333122 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Target criteria: Maximum likelihood

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