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- PDB-8wqp: Cryo-EM structure of T. pseudonana PyShell helical tube -

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Basic information

Entry
Database: PDB / ID: 8wqp
TitleCryo-EM structure of T. pseudonana PyShell helical tube
ComponentsDiatom the pyrenoid shell protein
KeywordsPLANT PROTEIN / marine diatoms / photosynthesis / CO2-concentrating mechanism / pyrenoids
Function / homologyDiatom pyrenoid shell protein / Uncharacterized protein
Function and homology information
Biological speciesThalassiosira pseudonana (Diatom)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKawamoto, A. / Tohda, R. / Gerle, C. / Kurisu, G.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23H04958 Japan
Japan Society for the Promotion of Science (JSPS)19H01153 Japan
Japan Science and TechnologyJPMJCR20E1 Japan
CitationJournal: Cell / Year: 2024
Title: Diatom pyrenoids are encased in a protein shell that enables efficient CO fixation.
Authors: Ginga Shimakawa / Manon Demulder / Serena Flori / Akihiro Kawamoto / Yoshinori Tsuji / Hermanus Nawaly / Atsuko Tanaka / Rei Tohda / Tadayoshi Ota / Hiroaki Matsui / Natsumi Morishima / ...Authors: Ginga Shimakawa / Manon Demulder / Serena Flori / Akihiro Kawamoto / Yoshinori Tsuji / Hermanus Nawaly / Atsuko Tanaka / Rei Tohda / Tadayoshi Ota / Hiroaki Matsui / Natsumi Morishima / Ryosuke Okubo / Wojciech Wietrzynski / Lorenz Lamm / Ricardo D Righetto / Clarisse Uwizeye / Benoit Gallet / Pierre-Henri Jouneau / Christoph Gerle / Genji Kurisu / Giovanni Finazzi / Benjamin D Engel / Yusuke Matsuda /
Abstract: Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO fixation. Diatoms fix up to 20% of global CO, but their pyrenoids remain poorly characterized. ...Pyrenoids are subcompartments of algal chloroplasts that increase the efficiency of Rubisco-driven CO fixation. Diatoms fix up to 20% of global CO, but their pyrenoids remain poorly characterized. Here, we used in vivo photo-crosslinking to identify pyrenoid shell (PyShell) proteins, which we localized to the pyrenoid periphery of model pennate and centric diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana. In situ cryo-electron tomography revealed that pyrenoids of both diatom species are encased in a lattice-like protein sheath. Single-particle cryo-EM yielded a 2.4-Å-resolution structure of an in vitro TpPyShell1 lattice, which showed how protein subunits interlock. T. pseudonana TpPyShell1/2 knockout mutants had no PyShell sheath, altered pyrenoid morphology, and a high-CO requiring phenotype, with reduced photosynthetic efficiency and impaired growth under standard atmospheric conditions. The structure and function of the diatom PyShell provide a molecular view of how CO is assimilated in the ocean, a critical ecosystem undergoing rapid change.
History
DepositionOct 12, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Nov 6, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Diatom the pyrenoid shell protein
B: Diatom the pyrenoid shell protein


Theoretical massNumber of molelcules
Total (without water)49,9722
Polymers49,9722
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Diatom the pyrenoid shell protein


Mass: 24986.020 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thalassiosira pseudonana (Diatom) / Gene: THAPSDRAFT_7881 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B8C7S8
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: in vitro tube structure of T. pseudonana PyShell / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thalassiosira pseudonana (Diatom)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
10.3 Msodium chlorideNaCl1
250 mMTris Hydrochloride AcidTris-HCl1
31 mMethylenediaminetetraacetic acidEDTA1
41 mMdithiothreitolDTT1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 500 nm / Cs: 0.01 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.631 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5951
Details: Images were collected in movie-mode at 52 frames per second
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Spherical aberration corrector: Microscope was modified with a Cs corrector

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10cryoSPARC4.0.2initial Euler assignment
11RELION4final Euler assignment
12RELION4classification
13RELION43D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -56.07 ° / Axial rise/subunit: 3.59 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 5951
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 322887 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023588
ELECTRON MICROSCOPYf_angle_d0.5054908
ELECTRON MICROSCOPYf_dihedral_angle_d4.266506
ELECTRON MICROSCOPYf_chiral_restr0.048556
ELECTRON MICROSCOPYf_plane_restr0.003662

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