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- PDB-7yk5: Rubisco from Phaeodactylum tricornutum bound to PYCO1(452-592) -

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Basic information

Entry
Database: PDB / ID: 7yk5
TitleRubisco from Phaeodactylum tricornutum bound to PYCO1(452-592)
Components
  • Multifunctional fusion protein
  • PYCO1 LSU binding motif
  • PYCO1 SSU binding motif
  • Ribulose bisphosphate carboxylase large chain
KeywordsPHOTOSYNTHESIS / Rubisco / phase separation / rubisco linker protein / condensation / pyrenoid / phaeodactylum tricornutum
Function / homology
Function and homology information


ribulose-bisphosphate carboxylase / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / chloroplast / monooxygenase activity / magnesium ion binding
Similarity search - Function
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, 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
Similarity search - Domain/homology
2-CARBOXYARABINITOL-1,5-DIPHOSPHATE / Multifunctional fusion protein / Ribulose bisphosphate carboxylase large chain
Similarity search - Component
Biological speciesPhaeodactylum tricornutum (Diatom)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 Å
AuthorsOh, Z.G. / Ang, W.S.L. / Bhushan, S. / Mueller-Cajar, O.
Funding support Singapore, 2items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore)MOE2018-T2-2-059 Singapore
Ministry of Education (MoE, Singapore)MOE2019-T3-1-012 Singapore
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs.
Authors: Zhen Guo Oh / Warren Shou Leong Ang / Cheng Wei Poh / Soak-Kuan Lai / Siu Kwan Sze / Hoi-Yeung Li / Shashi Bhushan / Tobias Wunder / Oliver Mueller-Cajar /
Abstract: The slow kinetics and poor substrate specificity of the key photosynthetic CO-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as ...The slow kinetics and poor substrate specificity of the key photosynthetic CO-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from . PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.
History
DepositionJul 21, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ribulose bisphosphate carboxylase large chain
B: Ribulose bisphosphate carboxylase large chain
C: Ribulose bisphosphate carboxylase large chain
D: Ribulose bisphosphate carboxylase large chain
E: Ribulose bisphosphate carboxylase large chain
F: Ribulose bisphosphate carboxylase large chain
G: Ribulose bisphosphate carboxylase large chain
H: Ribulose bisphosphate carboxylase large chain
I: Multifunctional fusion protein
J: Multifunctional fusion protein
K: Multifunctional fusion protein
L: Multifunctional fusion protein
M: Multifunctional fusion protein
N: Multifunctional fusion protein
O: Multifunctional fusion protein
P: Multifunctional fusion protein
i: PYCO1 SSU binding motif
k: PYCO1 SSU binding motif
j: PYCO1 SSU binding motif
l: PYCO1 SSU binding motif
h: PYCO1 LSU binding motif
g: PYCO1 LSU binding motif
f: PYCO1 LSU binding motif
e: PYCO1 LSU binding motif
d: PYCO1 LSU binding motif
c: PYCO1 LSU binding motif
b: PYCO1 LSU binding motif
a: PYCO1 LSU binding motif
hetero molecules


Theoretical massNumber of molelcules
Total (without water)576,56636
Polymers573,71728
Non-polymers2,8498
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, 550 kDa
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 54244.527 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Phaeodactylum tricornutum (Diatom) / Plasmid details: Pt 1 8.6 CCMP 2561
References: UniProt: E9PAI6, ribulose-bisphosphate carboxylase
#2: Protein
Multifunctional fusion protein


Mass: 16039.053 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Phaeodactylum tricornutum (Diatom) / Plasmid details: Pt 1 8.6 CCMP 2561 / References: UniProt: A0A6B9XNC0
#3: Protein/peptide
PYCO1 SSU binding motif


Mass: 875.973 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Pt 1 8.6 CCMP 2561 / Source: (gene. exp.) Phaeodactylum tricornutum (Diatom)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#4: Protein/peptide
PYCO1 LSU binding motif


Mass: 993.051 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: Pt 1 8.6 CCMP 2561 / Source: (gene. exp.) Phaeodactylum tricornutum (Diatom)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#5: Sugar
ChemComp-CAP / 2-CARBOXYARABINITOL-1,5-DIPHOSPHATE


Type: saccharideCarbohydrate / Mass: 356.115 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C6H14O13P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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 from Phaeodactylum tricornutum bound to linker protein PYCO1
Type: COMPLEX
Details: Rubisco purified from source organism, PYCO1 purified from E. coli.
Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 550 kDa/nm / Experimental value: YES
Source (natural)Organism: Phaeodactylum tricornutum (Diatom)
Buffer solutionpH: 8 / Details: 20 mM Tris pH 8.0 20 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisC4H11NO31
220 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 0.5 mg/mL Rubisco incubated with 21.9 uM of PYCO1(452-592).
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotted for 2 sec with blot force of 1.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 65 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8861
Details: Images were collected in movie mode at 10 frames per second.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Details: Gatan EF / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionLaplacian of gaussian
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 3354751
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 259796 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: Coot was used for model building
Atomic model buildingPDB-ID: 5MZ2

5mz2

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