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- PDB-8pcz: Ligand-free SpSLC9C1 in lipid nanodiscs, dimer -

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Basic information

Entry
Database: PDB / ID: 8pcz
TitleLigand-free SpSLC9C1 in lipid nanodiscs, dimer
ComponentsSperm-specific sodium proton exchanger
KeywordsMEMBRANE PROTEIN / SLC9 / NHE / sperm-specific
Function / homology
Function and homology information


potassium:proton antiporter activity / sperm head / sodium:proton antiporter activity / sodium ion import across plasma membrane / cGMP binding / single fertilization / sperm flagellum / cAMP binding / potassium ion transmembrane transport / cAMP-mediated signaling ...potassium:proton antiporter activity / sperm head / sodium:proton antiporter activity / sodium ion import across plasma membrane / cGMP binding / single fertilization / sperm flagellum / cAMP binding / potassium ion transmembrane transport / cAMP-mediated signaling / regulation of intracellular pH / protein homodimerization activity / plasma membrane
Similarity search - Function
Cation/H+ exchanger, CPA1 family / Cation/H+ exchanger / Sodium/hydrogen exchanger family / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Voltage-dependent channel domain superfamily / RmlC-like jelly roll fold
Similarity search - Domain/homology
Sperm-specific sodium:proton exchanger
Similarity search - Component
Biological speciesStrongylocentrotus purpuratus (purple sea urchin)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsKalienkova, V. / Peter, M. / Rheinberger, J. / Paulino, C.
Funding support Netherlands, Switzerland, 4items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
Swiss National Science FoundationP500PB_203053 Switzerland
Swiss National Science FoundationP2ZHP3_187679 Switzerland
CitationJournal: Nature / Year: 2023
Title: Structures of a sperm-specific solute carrier gated by voltage and cAMP.
Authors: Valeria Kalienkova / Martin F Peter / Jan Rheinberger / Cristina Paulino /
Abstract: The newly characterized sperm-specific Na/H exchanger stands out by its unique tripartite domain composition. It unites a classical solute carrier unit with regulatory domains usually found in ion ...The newly characterized sperm-specific Na/H exchanger stands out by its unique tripartite domain composition. It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain, which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals, our structure represents a potential new platform for the development of new on-demand contraceptives.
History
DepositionJun 11, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 8, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sperm-specific sodium proton exchanger
B: Sperm-specific sodium proton exchanger


Theoretical massNumber of molelcules
Total (without water)295,2492
Polymers295,2492
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sperm-specific sodium proton exchanger


Mass: 147624.484 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Strongylocentrotus purpuratus (purple sea urchin)
Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: A3RL54

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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: Ligand-free SpSLC9C1 in lipid nanodiscs, dimer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Strongylocentrotus purpuratus (purple sea urchin)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293S GnTI-
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2150 mMNaCl1
SpecimenConc.: 0.93 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288.15 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: 105000 X / Calibrated magnification: 59809 X / Nominal defocus max: 3000 nm / Nominal defocus min: 200 nm / Cs: 2.7 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: 2.51 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 11299
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9RELION3.1.0.initial Euler assignment
10RELION3.1.0.final Euler assignment
11RELION3.1.0.classification
12RELION3.1.0.3D reconstruction
13PHENIXmodel refinement
14ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5022107
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1458809 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316526
ELECTRON MICROSCOPYf_angle_d0.53322410
ELECTRON MICROSCOPYf_dihedral_angle_d4.2032188
ELECTRON MICROSCOPYf_chiral_restr0.0392694
ELECTRON MICROSCOPYf_plane_restr0.0042756

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