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- EMDB-45607: Anthoceros agrestis Rubisco octamer core complexed with Arabidops... -

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Basic information

Entry
Database: EMDB / ID: EMD-45607
TitleAnthoceros agrestis Rubisco octamer core complexed with Arabidopsis thaliana BSD2
Map data65 electrons over 50 movie frames super resolution mode. Ang/pix = 0.55. Rubisco 8 LSU with 8 BSD2 attached.
Sample
  • Complex: Rubisco assembled octamer core with Arabidopsis BSD2
    • Protein or peptide: Bundle Sheath Defective 2
    • Protein or peptide: Rubisco large subunit
KeywordsAnthoceros agrestis rubisco / PHOTOSYNTHESIS
Biological speciesAnthoceros agrestis (plant) / Arabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 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 2, 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_45607.png

Downloads & links

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Map

FileDownload / File: emd_45607.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation65 electrons over 50 movie frames super resolution mode. Ang/pix = 0.55. Rubisco 8 LSU with 8 BSD2 attached.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.55 Å/pix.
x 512 pix.
= 281.6 Å		0.55 Å/pix.
x 512 pix.
= 281.6 Å		0.55 Å/pix.
x 512 pix.
= 281.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.55 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045
Minimum - Maximum-0.06582823 - 0.20364703
Average (Standard dev.)0.0006462693 (±0.010487741)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_45607_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_45607_half_map_2.map
AnnotationHalf map B
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 octamer core with Arabidopsis BSD2

EntireName: Rubisco assembled octamer core with Arabidopsis BSD2
Components
  • Complex: Rubisco assembled octamer core with Arabidopsis BSD2
    • Protein or peptide: Bundle Sheath Defective 2
    • Protein or peptide: Rubisco large subunit

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Supramolecule #1: Rubisco assembled octamer core with Arabidopsis BSD2

SupramoleculeName: Rubisco assembled octamer core with Arabidopsis BSD2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Anthoceros agrestis (plant)
Molecular weightTheoretical: 490 KDa

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Macromolecule #1: Bundle Sheath Defective 2

MacromoleculeName: Bundle Sheath Defective 2 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 14.560366 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MANSLCFFSS PPTFCFQSPS KNPKPSHFFS TNDNTSSLVQ KRELLQTSRS QSFEVKAANN NPQGTKPNSL VCANCEGEGC VACSQCKGG GVNLIDHFNG QFKAGALCWL CRGKKEVLCG DCNGAGFIGG FLSTFDE

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Macromolecule #2: Rubisco large subunit

MacromoleculeName: Rubisco large subunit / type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
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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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
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
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: 79000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 522687
Startup modelType of model: OTHER
Details: Rubisco large subunit octamer core from previously solved pdb 9CHZ from an associated deposition. BSD2 was docked into the density from 8ILB, 8ILM and rigid-body refined.
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Software - details: Homogeneous refinement / Number images used: 33090
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final 3D classificationNumber classes: 4 / Avg.num./class: 183580 / Software - Name: cryoSPARC (ver. 4) / Software - details: 3D classification tool

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Atomic model buiding 1

Initial model
PDB IDChain

9chz

chain_id: all, source_name: PDB, initial_model_type: experimental model

8ilm

source_name: PDB, initial_model_type: experimental model

8ilb

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9ci1:
Anthoceros agrestis Rubisco octamer core complexed with Arabidopsis thaliana BSD2

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