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- PDB-9ci2: Anthoceros agrestis Rubisco octamer core complexed with small sub... -

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Basic information

Entry
Database: PDB / ID: 9ci2
TitleAnthoceros agrestis Rubisco octamer core complexed with small subunits and Arabidopsis thaliana BSD2
Components
  • Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic
  • Rubisco large subunit
  • Rubisco small subunit
KeywordsPHOTOSYNTHESIS / Anthoceros agrestis rubisco
Function / homology
Function and homology information


ribulose bisphosphate carboxylase complex assembly / protein folding chaperone complex / chloroplast stroma / : / protein folding chaperone / chloroplast / zinc ion binding / cytosol
Similarity search - Function
Heat shock protein DnaJ, cysteine-rich domain superfamily
Similarity search - Domain/homology
2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Anthoceros agrestis (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsAng, W.S.L. / Oh, Z.G. / Li, F.W. / Gunn, L.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB2213840 United States
National Science Foundation (NSF, United States)MCB2213841 United States
CitationJournal: Mol Plant / Year: 2024
Title: Unique biogenesis and kinetics of hornwort Rubiscos revealed by synthetic biology systems.
Authors: Zhen Guo Oh / Tanner Ashton Robison / Dan Hong Loh / Warren Shou Leong Ang / Jediael Zheng Ying Ng / Fay-Wei Li / Laura Helen Gunn /
Abstract: Hornworts are the only land plants that employ a pyrenoid to optimize Rubisco's CO fixation, yet hornwort Rubisco remains poorly characterized. Here we assembled the hornwort Anthoceros agrestis ...Hornworts are the only land plants that employ a pyrenoid to optimize Rubisco's CO fixation, yet hornwort Rubisco remains poorly characterized. Here we assembled the hornwort Anthoceros agrestis Rubisco (AaRubisco) using the Arabidopsis thaliana SynBio expression system and observed the formation of stalled intermediates, prompting us to develop a new SynBio system with A. agrestis cognate chaperones. We successfully assembled AaRubisco and Rubisco from three other hornwort species. Unlike A. thaliana Rubisco, AaRubisco assembly is not dependent on RbcX or Raf2. Kinetic characterization reveals that hornwort Rubiscos exhibit a range of catalytic rates (3-10 s), but with similar affinity (∼30 μM) and specificity (∼70) for CO. These results suggest that hornwort Rubiscos do not comply with the long-held canonical catalytic trade-off observed in other land plants, providing experimental support that Rubisco kinetics may be phylogenetically constrained. Unexpectedly, we observed a 50% increase in AaRubisco catalytic rates when RbcX was removed from our SynBio system, without any reduction in specificity. Structural biology, biochemistry, and proteomic analysis suggest that subtle differences in Rubisco large-subunit interactions, when RbcX is absent during biogenesis, increases the accessibility of active sites and catalytic turnover rate. Collectively, this study uncovered a previously unknown Rubisco kinetic parameter space and provides a SynBio chassis to expand the survey of other Rubisco kinetics. Our discoveries will contribute to developing new approaches for engineering Rubisco with superior kinetics.
History
DepositionJul 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic
2: Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic
3: Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic
4: Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic
A: Rubisco large subunit
B: Rubisco large subunit
C: Rubisco large subunit
D: Rubisco large subunit
E: Rubisco large subunit
F: Rubisco large subunit
G: Rubisco large subunit
H: Rubisco large subunit
I: Rubisco small subunit
J: Rubisco small subunit
K: Rubisco small subunit
L: Rubisco small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)541,86524
Polymers540,34316
Non-polymers1,5228
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Protein BUNDLE SHEATH DEFECTIVE 2, chloroplastic / AtBSD2


Mass: 14560.366 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: BSD2, At3g47650, F1P2.200 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9SN73
#2: Protein
Rubisco large subunit


Mass: 52908.777 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anthoceros agrestis (plant) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Protein
Rubisco small subunit


Mass: 14707.910 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anthoceros agrestis (plant) / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharide / Mass: 356.115 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C6H14O13P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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 assembly intermediate / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.51 MDa / Experimental value: YES
Source (natural)Organism: Anthoceros agrestis (plant)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8 / Details: 20 mM Tris pH 8.0 50 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
250 mMSodium chlorideNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 79000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4.8 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Details: Images were collected over 50 movie frames

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selectionBlob picker tool
2EPUimage acquisition
4cryoSPARC4CTF correctionPatch CTF estimation
7Coot0.9.8.1model fitting
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
11cryoSPARC4classification3D classification tool
12cryoSPARC43D reconstructionHomogeneous refinement
13PHENIX1.20.1-4487-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 522687
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19751 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-ID
19CHZ

9chz

all9CHZall1
28LIM

8lim

8LIM2
38ILB

8ilb

8ILB3

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