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- PDB-6z1g: CryoEM structure of the interaction between Rubisco Activase smal... -

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Entry
Database: PDB / ID: 6z1g
TitleCryoEM structure of the interaction between Rubisco Activase small-subunit-like (SSUL) domain with Rubisco from Nostoc sp. (strain PCC7120)
Components
  • Ribulose bisphosphate carboxylase large chain
  • Ribulose bisphosphate carboxylase small chain
  • Ribulose bisphosphate carboxylase/oxygenase activase
KeywordsPHOTOSYNTHESIS / 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
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: 8.2 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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Assembly

Deposited unit
A: Ribulose bisphosphate carboxylase/oxygenase activase
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase small chain
E: Ribulose bisphosphate carboxylase small chain


Theoretical massNumber of molelcules
Total (without water)142,1725
Polymers142,1725
Non-polymers00
Water0
1
A: Ribulose bisphosphate carboxylase/oxygenase activase
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase small chain
E: Ribulose bisphosphate carboxylase small chain

A: Ribulose bisphosphate carboxylase/oxygenase activase
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase small chain
E: Ribulose bisphosphate carboxylase small chain

A: Ribulose bisphosphate carboxylase/oxygenase activase
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase small chain
E: Ribulose bisphosphate carboxylase small chain

A: Ribulose bisphosphate carboxylase/oxygenase activase
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase small chain
E: Ribulose bisphosphate carboxylase small chain


  • defined by author
  • Evidence: native gel electrophoresis, The structure represents the repeat unit in an irregular network of Rca hexamers with Rubisco units. Rca forms a hexameric complex bearing six SSUL domains ...Evidence: native gel electrophoresis, The structure represents the repeat unit in an irregular network of Rca hexamers with Rubisco units. Rca forms a hexameric complex bearing six SSUL domains (residues 325-414), which bind to a cleft on the surface of Rubisco. Rubisco is a hexadecamer of eight RbcL and eight RbcS subunits. The Rubisco complex has D4 symmetry. The SSUL-(RbcL)2-(RbcS)2 repeat units can have one of two orientations (up or down). Thus Rubisco complexes saturated with SSUL domains can have four different configurations (uuuu, uuud, uudd, udud). In reality, some SSUL binding sites are probably left unoccupied. In addition, the AAA domains of Rca (residues 2-291) in the hexameric complex may interact with Rubisco, employing different contacts (related entry). The network is formed by flexible linkers connecting the SSUL domains in Rca, which then interlink Rubisco hexadecamers.
  • 569 kDa, 20 polymers
  • Search similar-shape structures of this assembly by Omokage search (details)
Theoretical massNumber of molelcules
Total (without water)568,68920
Polymers568,68920
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1
point symmetry operation3

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Components

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


Mass: 10266.549 Da / Num. of mol.: 1 / Fragment: SSUL 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 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P58555
#2: Protein Ribulose bisphosphate carboxylase large chain / RuBisCO large subunit


Mass: 53112.125 Da / Num. of mol.: 2
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.: 2
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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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
IDNameTypeEntity IDParent-IDSourceDetails
1Complex between Rubisco Activase small-subunit-like domain with Rubisco from Nostoc sp. (strain PCC7120)COMPLEXall0RECOMBINANT
2SSUL domain of Rca, part of the (Rca)6 hexamerCOMPLEX#11RECOMBINANT
3(RbcL)2(RbcS)2 unit of the (RbcL)8(RbcS)8 Rubisco complexCOMPLEX#2-#31RECOMBINANTThe (RbcL)8(RbcS)8 Rubisco complex has D4 symmetry. The (RbcL)2(RbcS)2 unit has two-fold roational symmetry.
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
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-IDStrainPlasmid
21Escherichia coli BL21 (bacteria)511693
32Escherichia coli BL21 (bacteria)511693BL21 STAR pBAD33-EcGroSELpHUE
43Escherichia coli BL21 (bacteria)511693BL21 STARpET11a-NosGroSEL-H6ubi-NosLS
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 mMDithiothreitolC4H10O2S21
SpecimenConc.: 4.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: For Preparing the NosRca:Rubisco complex, NosRubisco (1.25 uM) was mixed with NosRca (15 uM).
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 12 sec. / Electron dose: 47 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1570 / Details: 40 frames per image

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 298336
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32128 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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