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- PDB-8r34: CryoEM structure of the symmetric Pho90 dimer from yeast with sub... -

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Basic information

Entry
Database: PDB / ID: 8r34
TitleCryoEM structure of the symmetric Pho90 dimer from yeast with substrates.
ComponentsLow-affinity phosphate transporter PHO90
KeywordsMEMBRANE PROTEIN / Phosphate transporter / Plasma membrane protein
Function / homology
Function and homology information


regulation of phosphate transmembrane transport / polyphosphate metabolic process / phosphate transmembrane transporter activity / phosphate ion transport / cell periphery / transmembrane transport / plasma membrane
Similarity search - Function
Citrate transporter-like domain / Citrate transporter / SPX domain / SPX domain / SPX domain profile.
Similarity search - Domain/homology
1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE / PHOSPHATE ION / Low-affinity phosphate transporter PHO90
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.62 Å
AuthorsSchneider, S. / Kuehlbrandt, W. / Yildiz, O.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Structure / Year: 2024
Title: Complementary structures of the yeast phosphate transporter Pho90 provide insights into its transport mechanism.
Authors: Simon Schneider / Werner Kühlbrandt / Özkan Yildiz /
Abstract: Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined ...Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined the structures of the Saccharomyces cerevisiae phosphate importer Pho90 by electron cryomicroscopy in two complementary states at 2.3 and 3.1 Å resolution. The symmetrical, outward-open structure in the presence of phosphate indicates bound substrate ions in the binding pocket. In the absence of phosphate, Pho90 assumes an asymmetric structure with one monomer facing inward and one monomer facing outward, providing insights into the transport mechanism. The Pho90 transport domain binds phosphate ions on one side of the membrane, then flips to the other side where the substrate is released. Together with functional experiments, these complementary structures illustrate the transport mechanism of eukaryotic low-affinity phosphate transporters.
History
DepositionNov 8, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 24, 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: Low-affinity phosphate transporter PHO90
B: Low-affinity phosphate transporter PHO90
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,49416
Polymers195,5722
Non-polymers5,92214
Water1,11762
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15130 Å2
ΔGint-206 kcal/mol
Surface area35910 Å2

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Components

#1: Protein Low-affinity phosphate transporter PHO90


Mass: 97786.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PHO90, YJL198W, J0336 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P39535
#2: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical
ChemComp-3PH / 1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE / PHOSPHATIDIC ACID


Mass: 704.998 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C39H77O8P
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 62 / Source method: isolated from a natural source / Formula: H2O
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: Dimeric ScPho90 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMSodium chlorideNaCl1
220 mMPotassium phosphate1
SpecimenConc.: 3.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 defocus max: 2300 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 65 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192548 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037932
ELECTRON MICROSCOPYf_angle_d0.52510666
ELECTRON MICROSCOPYf_dihedral_angle_d12.8871351
ELECTRON MICROSCOPYf_chiral_restr0.0381263
ELECTRON MICROSCOPYf_plane_restr0.0041228

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