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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
DescriptorRibulose bisphosphate carboxylase large chain
Ribulose bisphosphate carboxylase small chain 1
KeywordsLYASE / beta barrel / lyase
Specimen sourceRhodobacter sphaeroides / archaea / ロドバクター・スファエロイデス
MethodElectron microscopy (3.39 Å resolution / Particle / Single particle)
AuthorsBracher, A. / Milicic, G. / Ciniawsky, S. / Wendler, P. / Hayer-Hartl, M. / Hartl, F.U.
CitationMol. Cell, 2017, 67, 744-756.e6

Mol. Cell, 2017, 67, 744-756.e6 Yorodumi Papers
Mechanism of Enzyme Repair by the AAA(+) Chaperone Rubisco Activase.
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

Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 3, 2017 / Release: Jul 26, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 26, 2017Structure modelrepositoryInitial release
1.1Aug 23, 2017Structure modelDatabase references / Refinement descriptioncitation / em_3d_fitting_citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _em_3d_fitting.target_criteria
1.2Sep 20, 2017Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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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
Polyers535,61716
Non-polymers3,04316
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)109360
ΔGint (kcal/M)-474
Surface area (Å2)126350
MethodPISA

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Components

#1: Polypeptide(L)
Ribulose bisphosphate carboxylase large chain / RuBisCO large subunit


Mass: 51768.852 Da / Num. of mol.: 8 / Fragment: RbcL
Source: (gene. exp.) Rhodobacter sphaeroides / archaea / ロドバクター・スファエロイデス
References: UniProt: P27997, EC: 4.1.1.39
#2: Polypeptide(L)
Ribulose bisphosphate carboxylase small chain 1 / RuBisCO small subunit 1


Mass: 15183.234 Da / Num. of mol.: 8
Source: (gene. exp.) Rhodobacter sphaeroides / archaea / ロドバクター・スファエロイデス
References: UniProt: P27998, EC: 4.1.1.39
#3: Chemical
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Mass: 356.115 Da / Num. of mol.: 8 / Formula: C6H14O13P2 / References: EC: 4.1.1.39
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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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
Source (recombinant)Organism: Escherichia coli BL21(DE3)
Buffer solutionpH: 8
Buffer component
IDConc.UnitsNameFormulaBuffer ID
120mMTRiS-HClC4H12ClNO31
250mMNaClNaCl1
31mMATPC10H16N5O13P31
41mMATP-gammaSC10H12Li4N5O12P3S1
51mMRuBPC5H12O11P21
SpecimenConc.: 0.3 mg/ml
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
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: 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
IDNameVersionCategoryImage processing IDFitting ID
4CTFFIND4CTF CORRECTION1
7Coot0.8.2MODEL FITTING1
9REFMAC5.8.0155MODEL REFINEMENT1
11RELIONFINAL EULER ASSIGNMENT1
12RELIONCLASSIFICATION1
13RELIONRECONSTRUCTION1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D4
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 333122 / Symmetry type: POINT
Atomic model buildingRef protocol: OTHER / Ref space: RECIPROCAL / Target criteria: Maximum likelihood

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