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- EMDB-45641: Anthoceros agrestis Rubisco assembled with RbcX1, RbcX2, Raf1, Ra... -

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Basic information

Entry
Database: EMDB / ID: EMD-45641
TitleAnthoceros agrestis Rubisco assembled with RbcX1, RbcX2, Raf1, Raf2 and BSD2
Map dataMain map
Sample
  • Complex: Rubisco assembled hexadecamer
    • Protein or peptide: RuBisCO large subunit
    • Protein or peptide: RuBisCO small subunit
  • Ligand: MAGNESIUM ION
  • Ligand: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE
KeywordsAnthoceros agrestis rubisco / PHOTOSYNTHESIS
Biological speciesAnthoceros agrestis (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsAng WSL / Oh ZG / Li FW / Gunn LH
Funding support United States, 2 items
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 8, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45641.png

Downloads & links

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Map

FileDownload / File: emd_45641.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 300 pix.
= 278.1 Å		0.93 Å/pix.
x 300 pix.
= 278.1 Å		0.93 Å/pix.
x 300 pix.
= 278.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.927 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0921
Minimum - Maximum-0.7614518 - 1.1661661
Average (Standard dev.)0.0051446934 (±0.06201632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 278.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_45641_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_45641_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rubisco assembled hexadecamer

EntireName: Rubisco assembled hexadecamer
Components
  • Complex: Rubisco assembled hexadecamer
    • Protein or peptide: RuBisCO large subunit
    • Protein or peptide: RuBisCO small subunit
  • Ligand: MAGNESIUM ION
  • Ligand: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

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Supramolecule #1: Rubisco assembled hexadecamer

SupramoleculeName: Rubisco assembled hexadecamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Anthoceros agrestis (plant)
Molecular weightTheoretical: 540 KDa

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Macromolecule #1: RuBisCO large subunit

MacromoleculeName: RuBisCO large subunit / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Anthoceros agrestis (plant)
Molecular weightTheoretical: 52.908777 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSPQTETKAG VGFKAGVKDY RLTYYTPDYE TKDTDILAAF RMTPQPGVPP EEAGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEP VAGEENQYIA YVAYPLDLFE EGSVTNMFTS IVGNVFGFKA LRALRLEDLR IPPAYSKTFQ GPPHGIQVER D KLNKYGRP ...String:
MSPQTETKAG VGFKAGVKDY RLTYYTPDYE TKDTDILAAF RMTPQPGVPP EEAGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEP VAGEENQYIA YVAYPLDLFE EGSVTNMFTS IVGNVFGFKA LRALRLEDLR IPPAYSKTFQ GPPHGIQVER D KLNKYGRP LLGCTIKPKL GLSAKNYGRA VYECLRGGLD FT(KCX)DDENVNS QPFMRWRDRF LFVAEAIFKS QAETGEIK G HYLNATAGTC EEMMKRAQFA RELGMPIVMH DYLTGGFTAN TTLAHYCRDN GLLLHIHRAM HAVIDRQRNH GIHFRVLAK ALRMSGGDHI HSGTVVGKLE GEREVTLGFV DLLRDDYIEK DRSRGIYFTQ DWVSMPGVLP VASGGIHVWH MPALTEIFGD DSVLQFGGG TLGHPWGNAP GAVANRVALE ACVQARNEGR DLAREGNDII REASKWSPEL AAACEVWKEI KFVFETIDTL

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Macromolecule #2: RuBisCO small subunit

MacromoleculeName: RuBisCO small subunit / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Anthoceros agrestis (plant)
Molecular weightTheoretical: 14.70791 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MQVWNPIDNP KFETLSYLPP LTDNQIAREI DYMLRNKWIP CLEFDPSGTI TTLPGQPGYY GGRYWTMWKL PMFGCNNAGY VLREIEHCK NAYPGCFIRV LGFDNIRQVQ CCAFIVHKPQ HHHHHH

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 8 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

MacromoleculeName: 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 8 / Formula: CAP
Molecular weightTheoretical: 356.115 Da
Chemical component information

ChemComp-CAP:
2-CARBOXYARABINITOL-1,5-DIPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
50.0 mMNaClSodium chloride

Details: 20 mM Tris pH 8.0 50 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
SoftwareName: EPU
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 4.8 sec. / Average electron dose: 65.0 e/Å2
Details: Images were collected over 50 movie frames in super resolution mode
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 63000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 761107
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1gk8


Details: Rigid body docking using 1GK8 as the starting model
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 87259
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Avg.num./class: 183580

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Atomic model buiding 1

Initial modelPDB ID:

1gk8


Chain - Chain ID: all / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9ck5:
Anthoceros agrestis Rubisco assembled with RbcX1, RbcX2, Raf1, Raf2 and BSD2

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