+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-12185 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM Structure of KdpFABC in E1 state with K | ||||||||||||
Map data | For model building, original map is tranformed by zflip | ||||||||||||
Sample |
| ||||||||||||
Function / homology | Function and homology information P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity ...P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / monoatomic cation transmembrane transport / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / magnesium ion binding / ATP hydrolysis activity / ATP binding / plasma membrane Similarity search - Function | ||||||||||||
Biological species | Escherichia coli K-12 (bacteria) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.38 Å | ||||||||||||
Authors | Sweet ME / Larsen C / Pedersen BP / Stokes DL | ||||||||||||
Funding support | United States, Denmark, 3 items
| ||||||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Structural basis for potassium transport in prokaryotes by KdpFABC. Authors: Marie E Sweet / Casper Larsen / Xihui Zhang / Michael Schlame / Bjørn P Pedersen / David L Stokes / Abstract: KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K ...KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K or water and which define a pathway for K transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K to the cytoplasm. | ||||||||||||
History |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_12185.map.gz | 118 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-12185-v30.xml emd-12185.xml | 27.1 KB 27.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_12185_fsc.xml | 11.2 KB | Display | FSC data file |
Images | emd_12185.png | 69.3 KB | ||
Masks | emd_12185_msk_1.map | 125 MB | Mask map | |
Others | emd_12185_half_map_1.map.gz emd_12185_half_map_2.map.gz | 115.9 MB 115.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-12185 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-12185 | HTTPS FTP |
-Related structure data
Related structure data | 7bh1MC 7bgyC 7bh2C 7lc3C 7lc6C M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_12185.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | For model building, original map is tranformed by zflip | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.035 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Mask #1
File | emd_12185_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half Map A from cryoSPARC non-linear refinement
File | emd_12185_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half Map A from cryoSPARC non-linear refinement | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half Map B from cryoSPARC non-linear refinement
File | emd_12185_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half Map B from cryoSPARC non-linear refinement | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : KdpFABC in E1 apo state
Entire | Name: KdpFABC in E1 apo state |
---|---|
Components |
|
-Supramolecule #1: KdpFABC in E1 apo state
Supramolecule | Name: KdpFABC in E1 apo state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 Details: KdpFABC complex solublized in decyl maltopyranoside (DM) detergent. |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: TK2499 / Recombinant plasmid: pSD107 |
Molecular weight | Theoretical: 160 KDa |
-Macromolecule #1: Potassium-transporting ATPase potassium-binding subunit
Macromolecule | Name: Potassium-transporting ATPase potassium-binding subunit type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 59.247684 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWRSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV ...String: MAAQGFLLIA TFLLVLMVLA RPLGSGLARL INDIPLPGTT GVERVLFRAL GVSDREMNWK QYLCAILGLN MLGLAVLFFM LLGQHYLPL NPQQLPGLSW DLALNTAVSF VTNTNWRSYS GETTLSYFSQ MAGLTVQNFL SAASGIAVIF ALIRAFTRQS M STLGNAWV DLLRITLWVL VPVALLIALF FIQQGALQNF LPYQAVNTVE GAQQLLPMGP VASQEAIKML GTNGGGFFNA NS SHPFENP TALTNFVQML AIFLIPTALC FAFGEVMGDR RQGRMLLWAM SVIFVICVGV VMWAEVQGNP HLLALGTDSS INM EGKESR FGVLVSSLFA VVTTAASCGA VIAMHDSFTA LGGMVPMWLM QIGEVVFGGV GSGLYGMMLF VLLAVFIAGL MIGR TPEYL GKKIDVREMK LTALAILVTP TLVLMGAALA MMTDAGRSAM LNPGPHGFSE VLYAVSSAAN NNGSAFAGLS ANSPF WNCL LAFCMFVGRF GVIIPVMAIA GSLVSKKSQA ASSGTLPTHG PLFVGLLIGT VLLVGALTFI PALALGPVAE YLS |
-Macromolecule #2: Potassium-transporting ATPase ATP-binding subunit
Macromolecule | Name: Potassium-transporting ATPase ATP-binding subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: P-type K+ transporter |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 72.251867 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G EAAPVIRE ...String: MSRKQLALFE PTLVVQALKE AVKKLNPQAQ WRNPVMFIVW IGSLLTTCIS IAMASGAMPG NALFSAAISG WLWITVLFAN FAEALAEGR SKAQANSLKG VKKTAFARKL REPKYGAAAD KVPADQLRKG DIVLVEAGDI IPCDGEVIEG GASVDESAIT G EAAPVIRE SGGDFASVTG GTRILSDWLV IECSVNPGET FLDRMIAMVE GAQRRKTPNE IALTILLIAL TIVFLLATAT LW PFSAWGG NAVSVTVLVA LLVCLIPTTI GGLLSAIGVA GMSRMLGANV IATSGRAVEA AGDVDVLLLD KTGTITLGNR QAS EFIPAQ GVDEKTLADA AQLASLADET PEGRSIVILA KQRFNLRERD VQSLHATFVP FTAQSRMSGI NIDNRMIRKG SVDA IRRHV EANGGHFPTD VDQKVDQVAR QGATPLVVVE GSRVLGVIAL KDIVKGGIKE RFAQLRKMGI KTVMITGDNR LTAAA IAAE AGVDDFLAEA TPEAKLALIR QYQAEGRLVA MTGDGTNDAP ALAQADVAVA MNSGTQAAKE AGNMVDLDSN PTKLIE VVH IGKQMLMTRG SLTTFSIAND VAKYFAIIPA AFAATYPQLN ALNIMCLHSP DSAILSAVIF NALIIVFLIP LALKGVS YK PLTASAMLRR NLWIYGLGGL LVPFIGIKVI DLLLTVCGLV |
-Macromolecule #3: Potassium-transporting ATPase KdpC subunit
Macromolecule | Name: Potassium-transporting ATPase KdpC subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 22.299225 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRPS ATAEMPYNPQ ASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE LVTASASGLD NNITPQAAAW QIPRVAKARN LSVEQLTQLI A KYSQQPLV ...String: MSGLRPALST FIFLLLITGG VYPLLTTVLG QWWFPWQANG SLIREGDTVR GSALIGQNFT GNGYFHGRPS ATAEMPYNPQ ASGGSNLAV SNPELDKLIA ARVAALRAAN PDASASVPVE LVTASASGLD NNITPQAAAW QIPRVAKARN LSVEQLTQLI A KYSQQPLV KYIGQPVVNI VELNLALDKL DEGTGLVPRG SSHHHHHHHH |
-Macromolecule #4: Potassium-transporting ATPase KdpF subunit
Macromolecule | Name: Potassium-transporting ATPase KdpF subunit / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Molecular weight | Theoretical: 3.071714 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MSAGVITGVL LVFLLLGYLV YALINAEAF |
-Macromolecule #5: POTASSIUM ION
Macromolecule | Name: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: K |
---|---|
Molecular weight | Theoretical: 39.098 Da |
-Macromolecule #6: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)...
Macromolecule | Name: (2R)-3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-2-(palmitoyloxy)propyl (E)-octadec-9-enoate type: ligand / ID: 6 / Number of copies: 1 / Formula: 9Y0 |
---|---|
Molecular weight | Theoretical: 717.996 Da |
Chemical component information | ChemComp-9Y0: |
-Macromolecule #7: water
Macromolecule | Name: water / type: ligand / ID: 7 / Number of copies: 6 / Formula: HOH |
---|---|
Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 4.5 mg/mL | |||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.5 Component:
| |||||||||||||||||||||
Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR Details: PELCO easiGlow Glow Discharge Cleaning System used for glow discharge with default settings. | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: 3 uL of protein mixture applied to grid. Blot time 4 seconds, blot force 0, no wait before plunging.. | |||||||||||||||||||||
Details | DM-solublized KdpFABC complex purified through SEC the same day as grid freezing. Peak fraction diluted to 4.5 mg/mL prior to application to grid. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Temperature | Min: 77.0 K / Max: 77.0 K |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2096 / Average exposure time: 10.0 sec. / Average electron dose: 50.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |