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- PDB-7snv: H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex -

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Basic information

Entry
Database: PDB / ID: 7snv
TitleH. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complex
Components
  • Carboxysome shell carbonic anhydrase
  • Ribulose bisphosphate carboxylase large chain
  • Ribulose bisphosphate carboxylase small chain
KeywordsLYASE / rubisco / TIM-barrel / complex
Function / homology
Function and homology information


carboxysome / ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / carbon fixation / reductive pentose-phosphate cycle / monooxygenase activity / carbonic anhydrase / carbonate dehydratase activity / magnesium ion binding / metal ion binding
Similarity search - Function
Carboxysome shell carbonic anhydrase / Carboxysome shell carbonic anhydrase, N-terminal / Carboxysome shell carbonic anhydrase, C-terminal / : / : / : / Carboxysome Shell Carbonic Anhydrase, C-terminal / Carboxysome Shell Carbonic Anhydrase, catalytic domain / Carboxysome Shell Carbonic Anhydrase, N-terminal / Ribulose bisphosphate carboxylase, small subunit ...Carboxysome shell carbonic anhydrase / Carboxysome shell carbonic anhydrase, N-terminal / Carboxysome shell carbonic anhydrase, C-terminal / : / : / : / Carboxysome Shell Carbonic Anhydrase, C-terminal / Carboxysome Shell Carbonic Anhydrase, catalytic domain / Carboxysome Shell Carbonic Anhydrase, N-terminal / 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 / Carboxysome shell carbonic anhydrase / Ribulose bisphosphate carboxylase small subunit
Similarity search - Component
Biological speciesHalothiobacillus neapolitanus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.07 Å
AuthorsBlikstad, C. / Dugan, E. / Laughlin, T.G. / Liu, M. / Shoemaker, S. / Remis, J. / Savage, D.F.
Funding support United States, 1items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-SC00016240 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Identification of a carbonic anhydrase-Rubisco complex within the alpha-carboxysome.
Authors: Cecilia Blikstad / Eli J Dugan / Thomas G Laughlin / Julia B Turnšek / Mira D Liu / Sophie R Shoemaker / Nikoleta Vogiatzi / Jonathan P Remis / David F Savage /
Abstract: Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO concentrating mechanism (CCM). In the ...Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO fixation-Rubisco and carbonic anhydrase-and are the centerpiece of the bacterial CO concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO by carbonic anhydrase, producing a high CO concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from . We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.
History
DepositionOct 28, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 3, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 1, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ribulose bisphosphate carboxylase large chain
B: Ribulose bisphosphate carboxylase small chain
C: Carboxysome shell carbonic anhydrase


Theoretical massNumber of molelcules
Total (without water)72,3893
Polymers72,3893
Non-polymers00
Water3,603200
1
A: Ribulose bisphosphate carboxylase large chain
B: Ribulose bisphosphate carboxylase small chain
C: Carboxysome shell carbonic anhydrase
x 8


Theoretical massNumber of molelcules
Total (without water)579,11024
Polymers579,11024
Non-polymers00
Water43224
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation7

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Components

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


Mass: 53831.723 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Strain: ATCC 23641 / c2 / Gene: cbbL, Hneap_0922 / Production host: Escherichia coli (E. coli)
References: UniProt: O85040, ribulose-bisphosphate carboxylase
#2: Protein Ribulose bisphosphate carboxylase small chain / RuBisCO small subunit


Mass: 12866.575 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Strain: ATCC 23641 / c2 / Gene: cbbS, rbcS, Hneap_0921 / Production host: Escherichia coli (E. coli)
References: UniProt: P45686, ribulose-bisphosphate carboxylase
#3: Protein/peptide Carboxysome shell carbonic anhydrase / CsoSCA / Carbonic anhydrase / CA / Carboxysome shell protein CsoS3


Mass: 5690.505 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
References: UniProt: O85042, carbonic anhydrase
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 200 / Source method: isolated from a natural source / Formula: H2O

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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-IDSource
1H. neapolitanus carboxysomal rubisco/CsoSCA-peptide (1-50)complexCOMPLEX#1-#30MULTIPLE SOURCES
2Ribulose bisphosphate carboxylase large chain (E.C.4.1.1.39), Ribulose bisphosphate carboxylase small chain (E.C.4.1.1.39)COMPLEX#1-#21RECOMBINANT
3Carboxysome shell carbonic anhydrase (E.C.4.2.1.1)COMPLEX#31SYNTHETIC
Molecular weightValue: 0.533 MDa / Experimental value: NO
Source (natural)Organism: Halothiobacillus neapolitanus (strain ATCC 23641 / c2) (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisC4H11NO31
280 mMsodium chlorideNaCl1
32 %glycerolC3H8O31
SpecimenConc.: 0.2665 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 0.5 uM of rubisco and 0.5 mM of peptide
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 seconds

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 57000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5742 / Details: 3 x 3 multishot image shift pattern.

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
7PHENIX1.19model fitting
9PHENIX1.19model refinement
13RELION3.13D reconstruction
CTF correctionDetails: CTF correction was performed during 3D reconstruction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52375 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Map-model FSC and geometry

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