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- EMDB-35158: Rubisco from Phaeodactylum tricornutum -

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Basic information

Entry
Database: EMDB / ID: EMD-35158
TitleRubisco from Phaeodactylum tricornutum
Map dataMain map of Rubisco from P. tricornutum
Sample
  • Complex: Ribulose 1,5-bisphosphate carboxylase/oxygenase from Phaeodactylum tricornutum
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain
KeywordsRubisco / phase separation / rubisco linker protein / condensation / pyrenoid / phaeodactylum tricornutum / PHOTOSYNTHESIS
Biological speciesPhaeodactylum tricornutum (Diatom)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.0 Å
AuthorsOh ZG / Ang WSL / Bhushan S / Mueller-Cajar O
Funding support Singapore, 2 items
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
DepositionJan 18, 2023-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJun 21, 2023-
Current statusJun 21, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35158.png

Downloads & links

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Map

FileDownload / File: emd_35158.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map of Rubisco from P. tricornutum
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 260 pix.
= 223.08 Å		0.86 Å/pix.
x 260 pix.
= 223.08 Å		0.86 Å/pix.
x 260 pix.
= 223.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.858 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.27330464 - 0.45201552
Average (Standard dev.)0.0009078689 (±0.020440247)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 223.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35158_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35158_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ribulose 1,5-bisphosphate carboxylase/oxygenase from Phaeodactylu...

EntireName: Ribulose 1,5-bisphosphate carboxylase/oxygenase from Phaeodactylum tricornutum
Components
  • Complex: Ribulose 1,5-bisphosphate carboxylase/oxygenase from Phaeodactylum tricornutum
    • Protein or peptide: Ribulose bisphosphate carboxylase large chain
    • Protein or peptide: Ribulose bisphosphate carboxylase small chain

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Supramolecule #1: Ribulose 1,5-bisphosphate carboxylase/oxygenase from Phaeodactylu...

SupramoleculeName: Ribulose 1,5-bisphosphate carboxylase/oxygenase from Phaeodactylum tricornutum
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Phaeodactylum tricornutum (Diatom) / Strain: Pt 1 8.6 CCMP 2561
Molecular weightTheoretical: 550 kDa/nm

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Macromolecule #1: Ribulose bisphosphate carboxylase large chain

MacromoleculeName: Ribulose bisphosphate carboxylase large chain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Phaeodactylum tricornutum (Diatom)
SequenceString: MSQSVSERTR IKSDRYESGV IPYAKMGYWD AAYAVKNTDV LALFRITPQP GVDPVEAAAA VAGESSTATW TVVWTDLLTA CDRYRAKAYR VDPVPNTTDQ YFAFIAYECD LFEEGSLANL TASIIGNVFG FKAVSALRLE DMRIPHSYLK TFQGPATGVI VERERLNKYG ...String:
MSQSVSERTR IKSDRYESGV IPYAKMGYWD AAYAVKNTDV LALFRITPQP GVDPVEAAAA VAGESSTATW TVVWTDLLTA CDRYRAKAYR VDPVPNTTDQ YFAFIAYECD LFEEGSLANL TASIIGNVFG FKAVSALRLE DMRIPHSYLK TFQGPATGVI VERERLNKYG IPLLGATVKP KLGLSGKNYG RVVYEGLKGG LDFLKDDENI NSQPFMRWRE RFLYCMEGIN RASAATGETK GSYLNITAGT MEEVYKRAEY AKTVGSIVVM IDLVMGYTAI QSAAIWARDN DLILHLHRAG NSTYARQKNH GINFRVICKW MRMCGVDHIH AGTVVGKLEG DPLMIKGFYD TLLLTHLNVN LPYGIFFEMT WASLRKCMPV ASGGIHCGQM HQLVHYLGDD VVLQFGGGTI GHPDGIQAGA TANRVALEAM ILARNEGADY FNSDIGPQIL RNAAKTCGPL QTALDLWKDI SFNYTSTDTS DFSVTPTANV

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Macromolecule #2: Ribulose bisphosphate carboxylase small chain

MacromoleculeName: Ribulose bisphosphate carboxylase small chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Phaeodactylum tricornutum (Diatom)
SequenceString:
MRLTQGCFSF LPDLTDQQIE KQIAYCITKG WAMNVEWTDD PHPRNSYWEL WGLPLFDVKD PASVMFELRE ARKSCAAGYI RINAFNAAYG TESCVMSFIV NRPSNEPGFY LERQELEGRR IAYTTKSYSV QANPEGGRY

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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 mMC5H11NO3Tris
20.0 mMNaClSodium chloride

Details: 20 mM Tris pH 8.0 20 mM NaCl
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.6 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted for 2 sec with blot force of 1..
Details0.5 mg/mL Rubisco

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV / Details: Gatan EF
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 8861 / Average exposure time: 5.0 sec. / Average electron dose: 65.0 e/Å2
Details: Images were collected in movie mode at 10 frames per second
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3354751
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5mz2


Details: Rigid body docking with Rubisco from similar species.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 256796
Initial angle assignmentType: COMMON LINE / Software - Name: RELION (ver. 3.1)
Details: Initial model was built in situ in Relion using Initial model generation
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 30 / Avg.num./class: 8659 / Software - Name: RELION (ver. 3.1) / Details: Final 2D classification used 259,796 particles.

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Atomic model buiding 1

DetailsCoot was used for model building
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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