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- PDB-6z1f: CryoEM structure of Rubisco Activase with its substrate Rubisco f... -

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Basic information

Entry
Database: PDB / ID: 6z1f
TitleCryoEM structure of Rubisco Activase with its substrate Rubisco from Nostoc sp. (strain PCC7120)
Components
  • (Ribulose bisphosphate carboxylase ...RuBisCO) x 2
  • Ribulose bisphosphate carboxylase/oxygenase activase
KeywordsCHAPERONE / AAA+ / beta barrel
Function / homology
Function and homology information


photorespiration / carboxysome / ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / monooxygenase activity / magnesium ion binding / ATP hydrolysis activity / ATP binding
Similarity search - Function
Ribulose bisphosphate carboxylase/oxygenase activase-like / : / Ribulose bisphosphate carboxylase/oxygenase activase, AAA, helical / 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/oxygenase activase-like / : / Ribulose bisphosphate carboxylase/oxygenase activase, AAA, helical / 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 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / Ribulose bisphosphate carboxylase large chain / Ribulose bisphosphate carboxylase small subunit / Ribulose bisphosphate carboxylase/oxygenase activase
Similarity search - Component
Biological speciesNostoc sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsWang, H. / Bracher, A. / Flecken, M. / Popilka, L. / Hartl, F.U. / Hayer-Hartl, M.
CitationJournal: Cell / Year: 2020
Title: Dual Functions of a Rubisco Activase in Metabolic Repair and Recruitment to Carboxysomes.
Authors: Mirkko Flecken / Huping Wang / Leonhard Popilka / F Ulrich Hartl / Andreas Bracher / Manajit Hayer-Hartl /
Abstract: Rubisco, the key enzyme of CO fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone ...Rubisco, the key enzyme of CO fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.
History
DepositionMay 13, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 23, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 7, 2021Group: Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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  • Deposited structure unit
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  • EMDB-11028
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Assembly

Deposited unit
1: Ribulose bisphosphate carboxylase/oxygenase activase
2: Ribulose bisphosphate carboxylase/oxygenase activase
3: Ribulose bisphosphate carboxylase/oxygenase activase
4: Ribulose bisphosphate carboxylase/oxygenase activase
5: Ribulose bisphosphate carboxylase/oxygenase activase
6: Ribulose bisphosphate carboxylase/oxygenase activase
A: Ribulose bisphosphate carboxylase large chain
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase large chain
E: Ribulose bisphosphate carboxylase large chain
F: Ribulose bisphosphate carboxylase large chain
G: Ribulose bisphosphate carboxylase large chain
H: Ribulose bisphosphate carboxylase large chain
I: Ribulose bisphosphate carboxylase small chain
J: Ribulose bisphosphate carboxylase small chain
K: Ribulose bisphosphate carboxylase small chain
L: Ribulose bisphosphate carboxylase small chain
M: Ribulose bisphosphate carboxylase small chain
N: Ribulose bisphosphate carboxylase small chain
O: Ribulose bisphosphate carboxylase small chain
P: Ribulose bisphosphate carboxylase small chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)730,43547
Polymers724,60722
Non-polymers5,82825
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Ribulose bisphosphate carboxylase ... , 2 types, 16 molecules ABCDEFGHIJKLMNOP

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


Mass: 53155.125 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Gene: cbbL, rbc, rbcA, rbcL, alr1524 / Plasmid: pET11a-NosGroSEL-H6ubi-NosLS / Cell line (production host): STAR / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P00879, ribulose-bisphosphate carboxylase
#3: Protein
Ribulose bisphosphate carboxylase small chain / RuBisCO small subunit


Mass: 12840.725 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Gene: cbbS, rbcS, alr1526 / Plasmid: pET11a-NosGroSEL-H6ubi-NosLS / Cell line (production host): STAR / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: P06514, ribulose-bisphosphate carboxylase

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Protein / Sugars , 2 types, 13 molecules 123456

#1: Protein
Ribulose bisphosphate carboxylase/oxygenase activase / RuBisCO activase


Mass: 32773.316 Da / Num. of mol.: 6 / Fragment: AAA+ domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nostoc sp. (strain PCC 7120 / SAG 25.82 / UTEX 2576) (bacteria)
Gene: rca, alr1533 / Plasmid: pHUE / Cell line (production host): pBAD33-EcGroSEL / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P58555
#7: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharideCarbohydrate / Mass: 356.115 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C6H14O13P2

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Non-polymers , 3 types, 18 molecules

#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1NosRca-deltaC:Rubisco complexCOMPLEXThe Rca hexamer captured in the complex with its substrate Rubisco from Nostoc sp. PCC 7120.#1-#30RECOMBINANT
2AAA-core of the Rca hexamer from Nostoc sp. PCC7120 in complex with ATP/ATPgammaSCOMPLEX#11RECOMBINANT
3Rubisco complex from Nostoc sp. PCC7120 in complex with inhibitor CABPCOMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11NO
210.1997 MDaYES
31NO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Nostoc sp. PCC 7120 = FACHB-418 (bacteria)103690
32Nostoc sp. PCC 7120 = FACHB-418 (bacteria)103690
43Nostoc sp. PCC 7120 = FACHB-418 (bacteria)103690
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
21Escherichia coli BL21 (bacteria)511693BL21 sTAR
32Escherichia coli BL21 (bacteria)511693BL21 sTAR
43Escherichia coli BL21 (bacteria)511693BL21 sTAR
Buffer solutionpH: 8.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMMalic acidC4H6O51
220 mM2-ethanesulfonic acidC6H13NO4S1
320 mM2-Amino-2-hydroxymethyl-propane-1,3-diolC4H11NO31
450 mMPotassium chlorideKCl1
510 mMMagnesium chlorideMgCl21
65 mMSodium bicarbonateNaHCO31
70.004 mM2-Carboxyarabinitol-1,5-diphosphateC6H14O13P21
82 mMAdenosine triphosphate sodium saltC10H16N5O13P31
92 mMAdenosine 5'-(3-thiotriphosphate) tetralithium saltC10H12Li4N5O12P3S1
SpecimenConc.: 2.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: NosRubisco (1 uM) was incubated with NaHCO3 (10 mM) at 298 K for 10 min followed by addition of CABP (8 uM). CABP-inhibited NosRubisco (0.5 uM) was then incubated with NosRcaDC (10 uM) in ...Details: NosRubisco (1 uM) was incubated with NaHCO3 (10 mM) at 298 K for 10 min followed by addition of CABP (8 uM). CABP-inhibited NosRubisco (0.5 uM) was then incubated with NosRcaDC (10 uM) in the presence of ATP (2 mM) for 10 s, followed by the addition of ATP-gammaS (2 mM), and incubated at 298 K for another 10 min before preparing the cryo-grids.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 2.8 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9042 / Details: 31 frames per image

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
13REFMAC5.8.0155model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 519151
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21149 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL

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