[English] 日本語
Yorodumi- EMDB-35491: Cryo-EM structure of non-gastric proton pump K794S mutant in Na+ ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-35491 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of non-gastric proton pump K794S mutant in Na+ bound E1AMPPCP state | |||||||||
Map data | ||||||||||
Sample |
| |||||||||
Keywords | P-type ATPase / P2-type ATPase / MEMBRANE PROTEIN | |||||||||
Biological species | Rattus (rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.65 Å | |||||||||
Authors | Abe K | |||||||||
Funding support | Japan, 1 items
| |||||||||
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 |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_35491.map.gz | 56.3 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-35491-v30.xml emd-35491.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_35491_fsc.xml | 8.2 KB | Display | FSC data file |
Images | emd_35491.png | 66 KB | ||
Masks | emd_35491_msk_1.map | 59.6 MB | Mask map | |
Others | emd_35491_half_map_1.map.gz emd_35491_half_map_2.map.gz | 55.3 MB 55.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-35491 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-35491 | HTTPS FTP |
-Validation report
Summary document | emd_35491_validation.pdf.gz | 853.5 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_35491_full_validation.pdf.gz | 853.1 KB | Display | |
Data in XML | emd_35491_validation.xml.gz | 15.7 KB | Display | |
Data in CIF | emd_35491_validation.cif.gz | 19.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-35491 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-35491 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_35491.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Voxel size | X=Y=Z: 1.328 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Mask #1
File | emd_35491_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: #1
File | emd_35491_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: #2
File | emd_35491_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : hetero dimer of alpha and beta subunit
Entire | Name: hetero dimer of alpha and beta subunit |
---|---|
Components |
|
-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 |
---|---|
Source (natural) | Organism: Rattus (rat) |
Molecular weight | Theoretical: 135 kDa/nm |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 8 mg/mL |
---|---|
Buffer | pH: 6.5 |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
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 |