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- EMDB-18049: Chlorella sorokiniana Rubisco: D4 symmetry imposed -

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Basic information

Entry
Database: EMDB / ID: EMD-18049
TitleChlorella sorokiniana Rubisco: D4 symmetry imposed
Map data
Sample
  • Complex: Rubisco holoenzyme purified from Chlorella sorokiniana UTEX1230
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small subunit, chloroplastic
  • Ligand: water
KeywordsRubisco / PLANT PROTEIN
Function / homology
Function and homology information


L-amino acid transmembrane transporter activity / photorespiration / ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / chloroplast / monooxygenase activity / magnesium ion binding / membrane
Similarity search - Function
Amino acid transporter, transmembrane domain / Transmembrane amino acid transporter protein / 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. ...Amino acid transporter, transmembrane domain / Transmembrane amino acid transporter protein / 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 / Thioredoxin / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
Ribulose bisphosphate carboxylase small subunit, chloroplastic / Ribulose bisphosphate carboxylase large chain
Similarity search - Component
Biological speciesChlorella sorokiniana (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsBarrett J / Blaza JN / Mackinder LCM
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research CouncilEP/W024063/1 United Kingdom
Future Leader FellowshipMR/T020679/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S015337/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011151/1a United Kingdom
Future Leader FellowshipMR/T040742/1 United Kingdom
CitationJournal: Nat Plants / Year: 2024
Title: A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage.
Authors: James Barrett / Mihris I S Naduthodi / Yuwei Mao / Clément Dégut / Sabina Musiał / Aidan Salter / Mark C Leake / Michael J Plevin / Alistair J McCormick / James N Blaza / Luke C M Mackinder /
Abstract: CO fixation is commonly limited by inefficiency of the CO-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO in phase-separated condensates called pyrenoids, which complete up to one- ...CO fixation is commonly limited by inefficiency of the CO-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO in phase-separated condensates called pyrenoids, which complete up to one-third of global CO fixation. Condensation of Rubisco in pyrenoids is dependent on interaction with disordered linker proteins that show little conservation between species. We developed a sequence-independent bioinformatic pipeline to identify linker proteins in green algae. We report the linker from Chlorella and demonstrate that it binds a conserved site on the Rubisco large subunit. We show that the Chlorella linker phase separates Chlamydomonas Rubisco and that despite their separation by ~800 million years of evolution, the Chlorella linker can support the formation of a functional pyrenoid in Chlamydomonas. This cross-species reactivity extends to plants, with the Chlorella linker able to drive condensation of some native plant Rubiscos in vitro and in planta. Our results represent an exciting frontier for pyrenoid engineering in plants, which is modelled to increase crop yields.
History
DepositionJul 27, 2023-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18049.png

Downloads & links

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Map

FileDownload / File: emd_18049.map.gz / Format: CCP4 / Size: 13.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 152 pix.
= 174.496 Å		1.15 Å/pix.
x 152 pix.
= 174.496 Å		1.15 Å/pix.
x 152 pix.
= 174.496 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.148 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.046
Minimum - Maximum-0.13395625 - 0.29444578
Average (Standard dev.)0.0012599995 (±0.019946173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions152152152
Spacing152152152
CellA=B=C: 174.496 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18049_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18049_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18049_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Rubisco holoenzyme purified from Chlorella sorokiniana UTEX1230

EntireName: Rubisco holoenzyme purified from Chlorella sorokiniana UTEX1230
Components
  • Complex: Rubisco holoenzyme purified from Chlorella sorokiniana UTEX1230
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small subunit, chloroplastic
  • Ligand: water

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Supramolecule #1: Rubisco holoenzyme purified from Chlorella sorokiniana UTEX1230

SupramoleculeName: Rubisco holoenzyme purified from Chlorella sorokiniana UTEX1230
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Chlorella sorokiniana (plant) / Strain: UTEX1230 (SAG211-8k) / Organelle: Pyrenoid / Location in cell: Chloroplast

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Macromolecule #1: Ribulose bisphosphate carboxylase large chain

MacromoleculeName: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Chlorella sorokiniana (plant) / Strain: UTEX1230 (SAG211-8k)
Molecular weightTheoretical: 52.580418 KDa
SequenceString: MAPQTETRAG AGFKAGVKDY RLTYYTPDYQ PKDTDILAAF RMTPQPGVPP EEAGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEP VPGEENQYIA YIAYPLDLFE EGSVTNLFTS IVGNVFGFKA LRALRLEDLR IPPAYVKTFQ GPPHGIQVER D KLNKYGRG ...String:
MAPQTETRAG AGFKAGVKDY RLTYYTPDYQ PKDTDILAAF RMTPQPGVPP EEAGAAVAAE SSTGTWTTVW TDGLTSLDRY KGRCYDIEP VPGEENQYIA YIAYPLDLFE EGSVTNLFTS IVGNVFGFKA LRALRLEDLR IPPAYVKTFQ GPPHGIQVER D KLNKYGRG LLGCTIKPKL GLSAKNYGRA VYECLRGGLD FTKDDENVNS QPFMRWRDRF LFVAEAIYKS QAETGEIKGH YL NATAATA EEMLKRAECA KDLGVPIIMH DYLTGGFTAN TSLAHYCRDN GLLLHIHRAM HAVIDRQRNH GIHFRVLAKA LRL SGGDHL HSGTVVGKLE GEREVTLGFV DLMRDDYIEK DRSRGIYFTQ DWVSLPGTMP VASGGIHVWH MPALVEIFGD DACL QFGGG TLGHPWGNAP GAAANRVALE ACTQARNEGR DLAREGGDVI RAACKWSPEL AAACEVWKEI KFEFETIDTL

UniProtKB: Ribulose bisphosphate carboxylase large chain

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Macromolecule #2: Ribulose bisphosphate carboxylase small subunit, chloroplastic

MacromoleculeName: Ribulose bisphosphate carboxylase small subunit, chloroplastic
type: protein_or_peptide / ID: 2 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Chlorella sorokiniana (plant) / Strain: UTEX1230 (SAG211-8k)
Molecular weightTheoretical: 20.507385 KDa
SequenceString:
MACTIAAVAP VAVRPVAATP LKQARNTFAA RTVSNATIKK TTAMQVWTPL NNKFFETFSY LPPMTDAEIS RQVDYIVSNG WTPCLEFAG AESAYTSNEN CVRMQNTTCL YYDNRYWTMW KLPMFGCTDG GQVLREVQAC RRAFPDAYIR VVGFDPVRQV Q VSGFLVNR PASVRDYQGP STRSV

UniProtKB: Ribulose bisphosphate carboxylase small subunit, chloroplastic

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 1255 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.3 mg/mL
BufferpH: 6.8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
200.0 mMC6H14O6Sorbitol
50.0 mMCH3CO2KPotassium acetate
2.0 mMMg(CH3COO)2,4H2OMagnesium acetate tetrahydrate
1.0 mMCaCl2Calcium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 11 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Details: Pressure was not recorded
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsChlorella sorokiniana Rubisco at 0.3 mg/mL (0.5 micromolar) was incubated with alpha3-alpha4 at 16 micromolar.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4447 / Average exposure time: 6.52 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 240000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 237035
Startup modelType of model: OTHER
Details: An initial model was created in Relion using particles from 2D classes.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 73962
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
Details: One round of 3D classification was completed following 2D classification. 73,962 particles were used for the reconstruction, representing one of the 3D classes.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial rigid body fitting was completed in UCSF Chimrea. Flexible fitting was completed in COOT and the coordinates were real-space refined in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 44
Output model

PDB-8q04:
Chlorella sorokiniana Rubisco: D4 symmetry imposed

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