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- PDB-9gtb: Cryo-EM structure of Mouse PMCA-NPTN complex captured in E1-Ca state -

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Basic information

Entry
Database: PDB / ID: 9gtb
TitleCryo-EM structure of Mouse PMCA-NPTN complex captured in E1-Ca state
Components
  • Neuroplastin
  • Plasma membrane calcium-transporting ATPase 2
KeywordsMEMBRANE PROTEIN / P-type Atpase / antiporter / calcium pump
Function / homology
Function and homology information


otolith mineralization / regulation of receptor localization to synapse / cerebellar Purkinje cell layer morphogenesis / type 1 fibroblast growth factor receptor binding / inner ear receptor cell differentiation / Reduction of cytosolic Ca++ levels / cGMP metabolic process / cerebellar granule cell differentiation / Ion transport by P-type ATPases / calcium-dependent ATPase activity ...otolith mineralization / regulation of receptor localization to synapse / cerebellar Purkinje cell layer morphogenesis / type 1 fibroblast growth factor receptor binding / inner ear receptor cell differentiation / Reduction of cytosolic Ca++ levels / cGMP metabolic process / cerebellar granule cell differentiation / Ion transport by P-type ATPases / calcium-dependent ATPase activity / excitatory synapse assembly / cerebellar Purkinje cell differentiation / positive regulation of fibroblast growth factor receptor signaling pathway / photoreceptor ribbon synapse / positive regulation of long-term neuronal synaptic plasticity / P-type Ca2+ transporter / detection of mechanical stimulus involved in sensory perception of sound / P-type calcium transporter activity / serotonin metabolic process / Ion homeostasis / positive regulation of calcium ion transport / locomotion / auditory receptor cell stereocilium organization / dendritic spine membrane / negative regulation of cytokine production / inner ear morphogenesis / neuromuscular process controlling balance / regulation of cell size / homophilic cell adhesion via plasma membrane adhesion molecules / inner ear development / cochlea development / immunological synapse / regulation of cytosolic calcium ion concentration / cell adhesion molecule binding / lactation / cerebellum development / PDZ domain binding / locomotory behavior / establishment of localization in cell / sensory perception of sound / synapse organization / positive regulation of neuron projection development / modulation of chemical synaptic transmission / neuron cellular homeostasis / regulation of synaptic plasticity / long-term synaptic potentiation / intracellular calcium ion homeostasis / positive regulation of protein phosphorylation / cell morphogenesis / neuron differentiation / calcium ion transport / positive regulation of cytosolic calcium ion concentration / presynaptic membrane / basolateral plasma membrane / calmodulin binding / neuron projection / postsynaptic density / cilium / apical plasma membrane / neuronal cell body / dendrite / calcium ion binding / glutamatergic synapse / endoplasmic reticulum / ATP hydrolysis activity / ATP binding / plasma membrane / cytoplasm
Similarity search - Function
Plasma membrane calcium transporting P-type ATPase, C-terminal / Plasma membrane calcium transporter ATPase C terminal / P-type ATPase, subfamily IIB / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / E1-E2 ATPase ...Plasma membrane calcium transporting P-type ATPase, C-terminal / Plasma membrane calcium transporter ATPase C terminal / P-type ATPase, subfamily IIB / Cation-transporting P-type ATPase, C-terminal / Cation transporting ATPase, C-terminus / Cation transporter/ATPase, N-terminus / Cation-transporting P-type ATPase, N-terminal / Cation transporter/ATPase, N-terminus / Cation transport ATPase (P-type) / E1-E2 ATPase / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N / E1-E2 ATPases phosphorylation site. / P-type ATPase, A domain superfamily / P-type ATPase / P-type ATPase, transmembrane domain superfamily / Immunoglobulin domain / haloacid dehalogenase-like hydrolase / Immunoglobulin I-set / Immunoglobulin I-set domain / HAD superfamily / HAD-like superfamily / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Chem-KXP / Neuroplastin / Plasma membrane calcium-transporting ATPase 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsVinayagam, D. / Raunser, S. / Sistel, O. / Shulte, U. / Constantin, C.E. / Prubaum, D. / Zolles, G. / Fakler, B.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
German Research Foundation (DFG)TRR 152/3, number 239283807 project P02 Germany
CitationJournal: Nature / Year: 2025
Title: Molecular mechanism of ultrafast transport by plasma membrane Ca-ATPases.
Authors: Deivanayagabarathy Vinayagam / Oleg Sitsel / Uwe Schulte / Cristina E Constantin / Wout Oosterheert / Daniel Prumbaum / Gerd Zolles / Bernd Fakler / Stefan Raunser /
Abstract: Tight control of intracellular Ca levels is fundamental as they are used to control numerous signal transduction pathways. Plasma membrane Ca-ATPases (PMCAs) have a crucial role in this process by ...Tight control of intracellular Ca levels is fundamental as they are used to control numerous signal transduction pathways. Plasma membrane Ca-ATPases (PMCAs) have a crucial role in this process by extruding Ca against a steep concentration gradient from the cytosol to the extracellular space. Although new details of PMCA biology are constantly being uncovered, the structural basis of the most distinguishing features of these pumps, namely, transport rates in the kilohertz range and regulation of activity by the plasma membrane phospholipid PtdIns(4,5)P, has so far remained elusive. Here we present the structures of mouse PMCA2 in the presence and absence of its accessory subunit neuroplastin in eight different stages of its transport cycle. Combined with whole-cell recordings that accurately track PMCA-mediated Ca extrusion in intact cells, these structures enable us to establish the first comprehensive transport model for a PMCA, reveal the role of disease-causing mutations and uncover the structural underpinnings of regulatory PMCA-phospholipid interaction. The transport cycle-dependent dynamics of PtdIns(4,5)P are fundamental for its role as a 'latch' promoting the fast release of Ca and opening a passageway for counter-ions. These actions are required for maintaining the ultra-fast transport cycle. Moreover, we identify the PtdIns(4,5)P-binding site as an unanticipated target for drug-mediated manipulation of intracellular Ca levels. Our work provides detailed structural insights into the uniquely fast operation of native PMCA-type Ca pumps and its control by membrane lipids and drugs.
History
DepositionSep 17, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Aug 6, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Sep 3, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Plasma membrane calcium-transporting ATPase 2
B: Neuroplastin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,50210
Polymers181,0872
Non-polymers2,4148
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Plasma membrane calcium-transporting ATPase 2 / PMCA2 / Plasma membrane calcium ATPase isoform 2 / Plasma membrane calcium pump isoform 2


Mass: 134647.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Atp2b2, Pmca2 / Cell line (production host): tsa201 / Production host: Homo sapiens (human) / References: UniProt: Q9R0K7, P-type Ca2+ transporter
#2: Protein Neuroplastin / Stromal cell-derived receptor 1 / SDR-1


Mass: 46440.059 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Nptn, Sdfr1, Sdr1 / Cell line (production host): tsA201 / Production host: Homo sapiens (human) / References: UniProt: P97300
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-KXP / (2S)-1-{[(R)-hydroxy{[(1R,2R,3S,4R,5R,6S)-2,3,6-trihydroxy-4,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl icosa-5,8,11,14-tetraenoate


Mass: 1047.088 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H85O19P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

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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: PMCA-NPTN complex / Type: COMPLEX
Details: prepared as separate cDNAs for transient (co)transfection of tsA201 nptn/basi double KO cells
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4 / Details: Tris 20mM NaCl 150mM
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The complex was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 55000 X / Calibrated magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 113 K / Temperature (min): 93 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 67.63 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 29014
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.7particle selection
2EPUimage acquisitionAFIS
4CTFFIND4CTF correction
7Coot9model fitting
9PHENIX1.21.1_5286model refinement
10cryoSPARC4initial Euler assignment
11cryoSPARC4final Euler assignment
12RELION3.1classification
13cryoSPARC43D reconstructionnon uniform refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 201573 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 146.2 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6A69

6a69


Accession code: 6A69 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.5 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610255
ELECTRON MICROSCOPYf_angle_d0.74413913
ELECTRON MICROSCOPYf_dihedral_angle_d8.041539
ELECTRON MICROSCOPYf_chiral_restr0.0471636
ELECTRON MICROSCOPYf_plane_restr0.0111757

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