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

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Basic information

Entry
Database: PDB / ID: 8q04
TitleChlorella sorokiniana Rubisco: D4 symmetry imposed
Components
  • Ribulose bisphosphate carboxylase large chain
  • Ribulose bisphosphate carboxylase small subunit, chloroplastic
KeywordsPLANT PROTEIN / Rubisco
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsBarrett, J. / Blaza, J.N. / Mackinder, L.C.M.
Funding support United Kingdom, 5items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
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 1.2Nov 27, 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.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ribulose bisphosphate carboxylase large chain
P: Ribulose bisphosphate carboxylase small subunit, chloroplastic
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase small subunit, chloroplastic
D: Ribulose bisphosphate carboxylase large chain
E: Ribulose bisphosphate carboxylase small subunit, chloroplastic
F: Ribulose bisphosphate carboxylase large chain
G: Ribulose bisphosphate carboxylase small subunit, chloroplastic
H: Ribulose bisphosphate carboxylase large chain
I: Ribulose bisphosphate carboxylase small subunit, chloroplastic
J: Ribulose bisphosphate carboxylase large chain
K: Ribulose bisphosphate carboxylase small subunit, chloroplastic
L: Ribulose bisphosphate carboxylase large chain
M: Ribulose bisphosphate carboxylase small subunit, chloroplastic
N: Ribulose bisphosphate carboxylase large chain
O: Ribulose bisphosphate carboxylase small subunit, chloroplastic


Theoretical massNumber of molelcules
Total (without water)584,70216
Polymers584,70216
Non-polymers00
Water22,6091255
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: native gel electrophoresis, The purified protein runs in a clean band well above a 240 kDa marker band.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Ribulose bisphosphate carboxylase large chain / RuBisCO large subunit


Mass: 52580.418 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Chlorella sorokiniana (plant) / Strain: UTEX1230 (SAG211-8k)
References: UniProt: W8SUA8, ribulose-bisphosphate carboxylase
#2: Protein
Ribulose bisphosphate carboxylase small subunit, chloroplastic


Mass: 20507.385 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Chlorella sorokiniana (plant) / Strain: UTEX1230 (SAG211-8k) / References: UniProt: A0A2P6U2H5
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1255 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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 holoenzyme purified from Chlorella sorokiniana UTEX1230
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Chlorella sorokiniana (plant) / Strain: UTEX1230 (SAG211-8k) / Cellular location: Chloroplast / Organelle: Pyrenoid
Buffer solutionpH: 6.8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2200 mMSorbitolC6H14O61
350 mMPotassium acetateCH3CO2K1
42 mMMagnesium acetate tetrahydrateMg(CH3COO)2,4H2O1
51 mMCalcium chlorideCaCl21
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Chlorella sorokiniana Rubisco at 0.3 mg/mL (0.5 micromolar) was incubated with alpha3-alpha4 at 16 micromolar.
Specimen supportDetails: Pressure was not recorded / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 240000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6.52 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4447

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionParticles were autopicked based on 2D classes following a round of manual picking
2EPUimage acquisitionIn AFIS mode
4CTFFIND4CTF correction
7UCSF Chimera1.17model fittingUsed for rigid body fitting
8Coot0.9.8.7model fittingUsed for flexible fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.20.1model refinementUsed for real-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 237035
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73962 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 44 / Protocol: FLEXIBLE FIT / Space: REAL
Details: Initial rigid body fitting was completed in UCSF Chimrea. Flexible fitting was completed in COOT and the coordinates were real-space refined in Phenix.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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