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Yorodumi- EMDB-35489: Cyo-EM structure of K794A non-gastric proton pump in Na+ bound E1... -
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-Basic information
Entry | Database: EMDB / ID: EMD-35489 | |||||||||
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Title | Cyo-EM structure of K794A non-gastric proton pump in Na+ bound E1AMPPCP state | |||||||||
Map data | ||||||||||
Sample |
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Keywords | P-type ATPase / P2-type ATPase / MEMBRANE PROTEIN | |||||||||
Function / homology | Function and homology information Basigin interactions / Ion transport by P-type ATPases / P-type potassium:proton transporter activity / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / metal ion transport / P-type sodium:potassium-exchanging transporter activity / membrane repolarization / sodium:potassium-exchanging ATPase complex / regulation of pH ...Basigin interactions / Ion transport by P-type ATPases / P-type potassium:proton transporter activity / positive regulation of sodium ion export across plasma membrane / positive regulation of potassium ion import across plasma membrane / metal ion transport / P-type sodium:potassium-exchanging transporter activity / membrane repolarization / sodium:potassium-exchanging ATPase complex / regulation of pH / regulation of calcium ion transmembrane transport / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / regulation of cardiac muscle contraction by calcium ion signaling / relaxation of cardiac muscle / organelle membrane / Ion homeostasis / sodium ion transport / potassium ion import across plasma membrane / monoatomic cation transmembrane transport / ATPase activator activity / blastocyst development / intercalated disc / lateral plasma membrane / sperm flagellum / sodium ion transmembrane transport / cardiac muscle contraction / ATP metabolic process / T-tubule / caveola / protein localization to plasma membrane / potassium ion transport / sarcolemma / intracellular calcium ion homeostasis / protein-macromolecule adaptor activity / ATPase binding / regulation of gene expression / basolateral plasma membrane / response to hypoxia / protein stabilization / cell adhesion / protein heterodimerization activity / apical plasma membrane / innate immune response / protein kinase binding / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / plasma membrane Similarity search - Function | |||||||||
Biological species | Rattus (rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.13 Å | |||||||||
Authors | Abe K | |||||||||
Funding support | Japan, 1 items
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Citation | Journal: Biochim Biophys Acta Mol Cell Res / Year: 2023 Title: An unusual conformation from Na-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na-binding. Authors: Kazuhiro Abe / Tomohiro Nishizawa / Pablo Artigas / Abstract: The Na,K-ATPase (NKA) and non-gastric H,K- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing ...The Na,K-ATPase (NKA) and non-gastric H,K- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na or H) and stoichiometries (3 Na:2 K or 1 H:1 K). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K occluded in E2-P and 3 Na-bound in E1·ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1·ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na, were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na at site II. Thus, the lack of Na at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na at site III induces the halfway rotation of TM6, which impairs Na-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na-binding in the NKA and other related cation pumps. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_35489.map.gz | 97.4 MB | EMDB map data format | |
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Header (meta data) | emd-35489-v30.xml emd-35489.xml | 16.9 KB 16.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_35489_fsc.xml | 9.8 KB | Display | FSC data file |
Images | emd_35489.png | 68.3 KB | ||
Masks | emd_35489_msk_1.map | 103 MB | Mask map | |
Others | emd_35489_half_map_1.map.gz emd_35489_half_map_2.map.gz | 95.6 MB 95.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-35489 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-35489 | HTTPS FTP |
-Related structure data
Related structure data | 8ijmMC 8ijlC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_35489.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Voxel size | X=Y=Z: 1.245 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_35489_msk_1.map | ||||||||||||
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Density Histograms |
-Half map: #1
File | emd_35489_half_map_1.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_35489_half_map_2.map | ||||||||||||
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Density Histograms |
-Sample components
-Entire : hetero dimer of alpha and beta subunit
Entire | Name: hetero dimer of alpha and beta subunit |
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Components |
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-Supramolecule #1: hetero dimer of alpha and beta subunit
Supramolecule | Name: hetero dimer of alpha and beta subunit / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
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Source (natural) | Organism: Rattus (rat) |
Molecular weight | Theoretical: 135 kDa/nm |
-Macromolecule #1: Sodium/potassium-transporting ATPase subunit alpha
Macromolecule | Name: Sodium/potassium-transporting ATPase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus (rat) |
Molecular weight | Theoretical: 109.084625 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: GMDLDDHRLS NTELEQKYGT NIIQGLSSVR ATELLARDGP NTLTPPKQTP EIIKFLKQMV GGFSILLWIG AALCWIAFVI QYVNNSASL DNVYLGAILV LVVILTGIFA YYQEAKSTNI MASFSKMIPQ QALVIRDAEK KVISAEQLVV GDVVEIKGGD Q IPADIRLV ...String: GMDLDDHRLS NTELEQKYGT NIIQGLSSVR ATELLARDGP NTLTPPKQTP EIIKFLKQMV GGFSILLWIG AALCWIAFVI QYVNNSASL DNVYLGAILV LVVILTGIFA YYQEAKSTNI MASFSKMIPQ QALVIRDAEK KVISAEQLVV GDVVEIKGGD Q IPADIRLV FSQGCKVDNS SLTGESEPQA RSTEFTHENP LETKNIGFYS TTCLEGTATG IVINTGDRTI IGRIASLASG VG SEKTPIA IEIEHFVHIV AGVAVSIDII FFITAVCMKY YVLDAIIFLI SIIVANVPEG LLATVTVTLS LTAKRMAKKN CLV KNLEAV ETLGSTSIIC SDKTGTLTQN RMTVAHLWFD NQIFVADTSE NQTKQAFDQS SGTWASLSKI ITLCNRAEFR PGQE SVPIM KRTVVGDASE TALLKFSEVI LGDVMGIRKR NHKVAEIPFN STNKFQLSIH ETEDPNNKRF LVVMKGAPER ILEKC STIM INGQEQPLDK SSADSFHTAY MELGGLGERV LGFCHLYLPA EQFPQSYIFD VDSVNFPTSN FCFVGLLSMI DPPRST VPD AVSKCRSAGI KVIMVTGDHP ITAKAIAKSV GIISANNETV EDIAKRRNIA VEQVNKREAK AAVVTGMELK DMTPEQL DE LLTNYQEIVF ARTSPQQKLI IVEGCQRQDA IVAVTGDGVN DSPALKKADI GIAMGIAGSD AAKNAADMVL LDDNFASI V TGVEEGRLIF DNLKKTIAYT LTANIAELCP FLIYIVAGLP LPIGTITILF IDLGTDIIPS IALAYEKAES DIMNRKPRH KKKDRLVNTQ LAIYSYLHIG LMQALGGFLV YFTVYAQQGF WPTSLINLRV AWETDDINDL EDSYGQEWTR YQRKYLEWTG STAFFVAIM IQQIADLIIR KTRRNSIFQQ GLFRNKVIWV GIASQVIVAL ILSYGLGSVP ALSFTMLRVQ YWFVAVPHAI L IWVYDEMR KLFIRLYPGS WWDKNMYY UniProtKB: Sodium/potassium-transporting ATPase subunit alpha |
-Macromolecule #2: Sodium/potassium-transporting ATPase subunit beta-1
Macromolecule | Name: Sodium/potassium-transporting ATPase subunit beta-1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus (rat) |
Molecular weight | Theoretical: 37.516859 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MGDYKDDDDK SSGENLYFQG MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIQVMLLT ISELKPTYQ DRVAPPGLTQ IPQIQKTEIS FRPNDPKSYE AYVLNIIRFL EKYKDSAQKD DMIFEDCGSM PSEPKERGEF N HERGERKV ...String: MGDYKDDDDK SSGENLYFQG MARGKAKEEG SWKKFIWNSE KKEFLGRTGG SWFKILLFYV IFYGCLAGIF IGTIQVMLLT ISELKPTYQ DRVAPPGLTQ IPQIQKTEIS FRPNDPKSYE AYVLNIIRFL EKYKDSAQKD DMIFEDCGSM PSEPKERGEF N HERGERKV CRFKLDWLGN CSGLNDESYG YKEGKPCIII KLNRVLGFKP KPPKNESLET YPLTMKYNPN VLPVQCTGKR DE DKDKVGN IEYFGMGGFY GFPLQYYPYY GKLLQPKYLQ PLLAVQFTNL TLDTEIRIEC KAYGENIGYS EKDRFQGRFD VKI EVKS UniProtKB: Sodium/potassium-transporting ATPase subunit beta-1 |
-Macromolecule #3: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
Macromolecule | Name: PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 1 / Formula: ACP |
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Molecular weight | Theoretical: 505.208 Da |
Chemical component information | ChemComp-ACP: |
-Macromolecule #4: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
Macromolecule | Name: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PCW |
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Molecular weight | Theoretical: 787.121 Da |
Chemical component information | ChemComp-PCW: |
-Macromolecule #5: CHOLESTEROL
Macromolecule | Name: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 1 / Formula: CLR |
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Molecular weight | Theoretical: 386.654 Da |
Chemical component information | ChemComp-CLR: |
-Macromolecule #6: SODIUM ION
Macromolecule | Name: SODIUM ION / type: ligand / ID: 6 / Number of copies: 2 |
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Molecular weight | Theoretical: 22.99 Da |
-Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 1 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 8 mg/mL |
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Buffer | pH: 6.5 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 53.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |