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- PDB-9gsy: Cryo-EM structure of mouse PMCA captured in E2-P state (BEF3) -

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Basic information

Entry
Database: PDB / ID: 9gsy
TitleCryo-EM structure of mouse PMCA captured in E2-P state (BEF3)
ComponentsPlasma membrane calcium-transporting ATPase 2
KeywordsMEMBRANE PROTEIN / Calcium pump / Ptype-Atpase
Function / homology
Function and homology information


otolith mineralization / cerebellar Purkinje cell layer morphogenesis / inner ear receptor cell differentiation / Reduction of cytosolic Ca++ levels / cGMP metabolic process / Ion transport by P-type ATPases / cerebellar granule cell differentiation / calcium-dependent ATPase activity / cerebellar Purkinje cell differentiation / photoreceptor ribbon synapse ...otolith mineralization / cerebellar Purkinje cell layer morphogenesis / inner ear receptor cell differentiation / Reduction of cytosolic Ca++ levels / cGMP metabolic process / Ion transport by P-type ATPases / cerebellar granule cell differentiation / calcium-dependent ATPase activity / cerebellar Purkinje cell differentiation / photoreceptor ribbon synapse / P-type Ca2+ transporter / P-type calcium transporter activity / detection of mechanical stimulus involved in sensory perception of sound / serotonin metabolic process / Ion homeostasis / positive regulation of calcium ion transport / locomotion / auditory receptor cell stereocilium organization / dendritic spine membrane / inner ear morphogenesis / neuromuscular process controlling balance / regulation of cell size / inner ear development / cochlea development / regulation of cytosolic calcium ion concentration / lactation / cerebellum development / PDZ domain binding / locomotory behavior / establishment of localization in cell / sensory perception of sound / synapse organization / neuron cellular homeostasis / regulation of synaptic plasticity / cell morphogenesis / neuron differentiation / intracellular calcium ion homeostasis / calcium ion transport / presynaptic membrane / basolateral plasma membrane / calmodulin binding / neuron projection / cilium / apical plasma membrane / neuronal cell body / 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 / haloacid dehalogenase-like hydrolase / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
BERYLLIUM TRIFLUORIDE ION / Chem-KXP / Plasma membrane calcium-transporting ATPase 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsVinayagam, D. / Raunser, S. / Sistel, O. / Schulte, U. / Constantin, C.E. / Prumbaum, 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 16, 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
Revision 1.0Aug 6, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 6, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)135,7854
Polymers134,6471
Non-polymers1,1373
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: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: BeF3
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#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
Has ligand of interestY
Has protein modificationN

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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: mouse PMCA captured in E2-P state (BEF3) / Type: COMPLEX
Details: prepared as separate cDNAs for transient (co)transfection of tsA201 nptn/basi double KO cells
Entity ID: #1 / 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.: 2 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: 3.5 sec. / Electron dose: 57 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 13134
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
9cryoSPARC4initial Euler assignment
10cryoSPARC4final Euler assignment
11RELION3.1classification
12cryoSPARC43D reconstructionnon uniform refinement
13PHENIX1.21.1_5286model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 437154 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 181.1 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 6A69

6a69


Accession code: 6A69 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.52 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037484
ELECTRON MICROSCOPYf_angle_d0.49610146
ELECTRON MICROSCOPYf_dihedral_angle_d12.8172796
ELECTRON MICROSCOPYf_chiral_restr0.0411202
ELECTRON MICROSCOPYf_plane_restr0.0041277

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