+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-35159 | |||||||||
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Title | PYCO1(452-592) from Phaeodactylum tricornutum | |||||||||
Map data | PYCO1(452-592) | |||||||||
Sample |
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Keywords | Rubisco / phase separation / rubisco linker protein / condensation / pyrenoid / phaeodactylum tricornutum / PHOTOSYNTHESIS | |||||||||
Biological species | Phaeodactylum tricornutum (Diatom) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.6 Å | |||||||||
Authors | Oh ZG / Ang WSL / Bhushan S / Mueller-Cajar O | |||||||||
Funding support | Singapore, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_35159.map.gz | 84.6 KB | EMDB map data format | |
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Header (meta data) | emd-35159-v30.xml emd-35159.xml | 19.4 KB 19.4 KB | Display Display | EMDB header |
Images | emd_35159.png | 55.2 KB | ||
Others | emd_35159_half_map_1.map.gz emd_35159_half_map_2.map.gz | 37.5 MB 37.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-35159 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-35159 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_35159.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | PYCO1(452-592) | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.858 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: non-masked half map
File | emd_35159_half_map_1.map | ||||||||||||
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Annotation | non-masked half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: non-masked half map
File | emd_35159_half_map_2.map | ||||||||||||
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Annotation | non-masked half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : PYCO1 bound to Rubisco from Phaeodactylum tricornutum
Entire | Name: PYCO1 bound to Rubisco from Phaeodactylum tricornutum |
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Components |
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-Supramolecule #1: PYCO1 bound to Rubisco from Phaeodactylum tricornutum
Supramolecule | Name: PYCO1 bound to Rubisco from Phaeodactylum tricornutum / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Phaeodactylum tricornutum (Diatom) / Strain: Pt 1 8.6 CCMP 2561 |
Molecular weight | Theoretical: 60 kDa/nm |
-Macromolecule #1: Pyrenoid Component 1 (452-592)
Macromolecule | Name: Pyrenoid Component 1 (452-592) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Phaeodactylum tricornutum (Diatom) |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: KWSPRGGS |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.38 mg/mL | |||||||||
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Buffer | pH: 8 Component:
Details: 20 mM Tris pH 8.0 20 mM NaCl | |||||||||
Grid | Model: 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 | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted for 2 sec with blot force of 1.. | |||||||||
Details | 0.38 mg/mL PYCO1(452-592) with 0.5 mg/mL Phaeodactylum tricornutum rubisco |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000 |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV / Details: Gatan EF |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film 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 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 3354751 |
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Startup model | Type of model: NONE / Details: De novo model was built into the density map. |
Initial angle assignment | Type: COMMON LINE / Software - Name: RELION (ver. 3.1) Details: Initial model was built in situ in Relion using Initial model generation |
Final 3D classification | Number classes: 30 / Avg.num./class: 8659 / Software - Name: RELION (ver. 3.1) / Details: Final 2D classification used 259,796 particles. |
Final angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 3.1) |
Final reconstruction | Number classes used: 1 / Applied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 259796 |
-Atomic model buiding 1
Details | Coot was used for model building |
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Refinement | Space: REAL / Protocol: AB INITIO MODEL |