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- PDB-9chz: Anthoceros agrestis Rubisco assembled with Raf1 Raf2 and BSD2 -

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Basic information

Entry
Database: PDB / ID: 9chz
TitleAnthoceros agrestis Rubisco assembled with Raf1 Raf2 and BSD2
Components
  • Rubisco large subunit
  • Rubisco small subunit
KeywordsPHOTOSYNTHESIS / Anthoceros agrestis rubisco
Function / homology2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
Function and homology information
Biological speciesAnthoceros 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 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 assemble 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 assemble 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. In other words, 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. This study uncovered a previously unknown Rubisco kinetic parameter space and provides a SynBio chassis to expand the survey of other Rubisco kinetics. Our discovery could thus reshape the 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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
M: Rubisco small subunit
N: Rubisco small subunit
O: Rubisco small subunit
P: Rubisco small subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)543,97732
Polymers540,93316
Non-polymers3,04316
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: 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-Gold(DE3)pLysS AG' (bacteria)
#2: Protein
Rubisco small subunit


Mass: 14707.910 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anthoceros agrestis (plant)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharide / Mass: 356.115 Da / Num. of mol.: 8 / 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 assembled hexadecamer / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.54 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 mesh size: 400 divisions/in. / 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: 63000 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: 3252384
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 222666 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 1GK8

1gk8


Pdb chain-ID: all / Accession code: 1GK8 / Source name: PDB / Type: experimental model

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