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- PDB-7nnp: Rb-loaded cryo-EM structure of the E1-ATP KdpFABC complex. -

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Basic information

Entry
Database: PDB / ID: 7nnp
TitleRb-loaded cryo-EM structure of the E1-ATP KdpFABC complex.
Components(Potassium-transporting ATPase ...) x 4
KeywordsMEMBRANE PROTEIN / P-type ATPase / superfamily of K+ transporters (SKT) / Rb substitution / intersubunit tunnel
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 / plasma membrane => GO:0005886 / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / hydrolase activity ...P-type K+ transporter / potassium:proton antiporter complex / potassium ion-transporting ATPase complex / P-type potassium transmembrane transporter activity / monoatomic cation transmembrane transport / plasma membrane => GO:0005886 / potassium ion binding / potassium ion transmembrane transport / potassium ion transport / hydrolase activity / magnesium ion binding / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / P-type ATPase, haloacid dehalogenase domain / P-type ATPase, phosphorylation site / P-type ATPase, cytoplasmic domain N ...Potassium-transporting ATPase C chain / Potassium-transporting ATPase A chain / K+ transporting P-type ATPase, F subunit / K+-transporting ATPase, c chain / Potassium-transporting ATPase A subunit / F subunit of K+-transporting ATPase (Potass_KdpF) / P-type ATPase, B chain, subfamily IA / 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 / HAD superfamily / HAD-like superfamily
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / CARDIOLIPIN / RUBIDIUM ION / Potassium-transporting ATPase ATP-binding subunit / Potassium-transporting ATPase KdpC subunit / Potassium-transporting ATPase potassium-binding subunit / Potassium-transporting ATPase KdpF subunit
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSilberberg, J.M. / Corey, R.A. / Hielkema, L. / Stock, C. / Stansfeld, P.J. / Paulino, C. / Haenelt, I.
Funding support Germany, Netherlands, United Kingdom, 5items
OrganizationGrant numberCountry
German Research Foundation (DFG)8A6322/3 Germany
German Research Foundation (DFG)CRC807 Germany
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
Wellcome Trust208361/Z/17/Z United Kingdom
Citation
Journal: Nat Commun / Year: 2021
Title: Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC.
Authors: Jakob M Silberberg / Robin A Corey / Lisa Hielkema / Charlott Stock / Phillip J Stansfeld / Cristina Paulino / Inga Hänelt /
Abstract: KdpFABC, a high-affinity K pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD ...KdpFABC, a high-affinity K pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.
#1: Journal: Biorxiv / Year: 2021
Title: Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC
Authors: Silberberg, J.M. / Corey, R.A. / Hielkema, L. / Stock, C. / Stansfeld, P.J. / Paulino, C. / Hanelt, I.
History
DepositionFeb 25, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / em_admin / pdbx_database_proc
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Potassium-transporting ATPase potassium-binding subunit
C: Potassium-transporting ATPase KdpC subunit
D: Potassium-transporting ATPase KdpF subunit
B: Potassium-transporting ATPase ATP-binding subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)158,78015
Polymers154,6634
Non-polymers4,11711
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21370 Å2
ΔGint-264 kcal/mol
Surface area50110 Å2
MethodPISA

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Components

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Potassium-transporting ATPase ... , 4 types, 4 molecules ACDB

#1: Protein Potassium-transporting ATPase potassium-binding subunit / ATP phosphohydrolase [potassium-transporting] A chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] A chain / Potassium-binding and translocating subunit A / Potassium-translocating ATPase A chain


Mass: 59276.652 Da / Num. of mol.: 1 / Mutation: G232D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: kdpA, D9J52_12825, SAMEA3752559_01047 / Production host: Escherichia coli (E. coli) / Strain (production host): LB2003 / References: UniProt: A0A2S5ZPF1
#2: Protein Potassium-transporting ATPase KdpC subunit / ATP phosphohydrolase [potassium-transporting] C chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] C chain / Potassium-binding and translocating subunit C / Potassium-translocating ATPase C chain


Mass: 20281.035 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: kdpC, A9R57_11470, ACU57_12815, AM464_20755, AUQ13_21370, BANRA_01742, BANRA_03870, BHS87_03640, BJJ90_18865, BMA87_25495, BON98_02815, BUE81_20120, BvCms12BK_01599, BvCms2454_04067, ...Gene: kdpC, A9R57_11470, ACU57_12815, AM464_20755, AUQ13_21370, BANRA_01742, BANRA_03870, BHS87_03640, BJJ90_18865, BMA87_25495, BON98_02815, BUE81_20120, BvCms12BK_01599, BvCms2454_04067, BvCmsHHP001_00774, BvCmsHHP056_03395, BvCmsKSP026_01797, BW690_07920, C5F72_18220, C5F73_11285, C5N07_14650, C9E25_26865, CA593_25675, CG692_12945, CI694_20645, D0X26_17510, D2185_15885, D3821_21865, D3Y67_05220, D4718_15195, D9C99_19315, D9D20_12860, D9D44_15890, D9G69_25005, D9J52_12835, D9Z28_23025, DAH34_18920, DBQ99_18020, DJ503_06890, DL326_22940, DNC98_20170, DT034_24065, DTL43_19970, DXT73_03320, E2119_26465, E2134_18585, E2135_05100, E4K51_13735, E4K53_14000, E4K55_14005, E4K61_12665, E5P28_21685, EA213_10645, EAI52_22450, EC3234A_8c00230, ECTO6_03352, ED307_21640, EEP23_23705, EG808_19380, EI021_12890, EI028_12880, EI032_09320, EI041_12810, EIZ93_03125, EL75_3091, EL79_3184, EL80_5457, ERS085365_04786, ERS085416_02369, ERS139211_04506, ERS150873_01994, EYD11_15990, EYV18_15885, F1E19_24880, F9040_19740, FNW97_03505, FRV13_05295, FV293_12595, G5688_17415, GII66_07600, GKF39_08170, GKF74_06650, GKF86_08095, GKF89_05805, GP689_15935, GP954_16115, GP979_23630, GQE33_23970, GQE34_25250, GQE64_10530, GQM06_27255, GQM17_23755, GRW05_21650, GRW42_11025, GRW57_15570, GRW80_11135, GRW81_16520, HVY93_16490, HX136_18175, NCTC10963_03490, NCTC8500_03902, NCTC9045_04050, NCTC9058_03392, NCTC9062_04759, NCTC9073_02588, NCTC9706_00826, PGD_02635, RK56_025175, SAMEA3472044_02479, SAMEA3472047_02083, SAMEA3472080_00272, SAMEA3484427_03080, SAMEA3484429_03302, SAMEA3753097_03043, SAMEA3753300_04144, WP2S18E08_32510, WP7S17E04_30600
Production host: Escherichia coli (E. coli) / Strain (production host): LB2003 / References: UniProt: A0A037YI39
#3: Protein/peptide Potassium-transporting ATPase KdpF subunit / ATP phosphohydrolase [potassium-transporting] F chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] F chain / Potassium-binding and translocating subunit F / Potassium-translocating ATPase F chain


Mass: 2853.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: kdpF, b4513, JW0687 / Production host: Escherichia coli (E. coli) / Strain (production host): LB2003 / References: UniProt: P36937
#4: Protein Potassium-transporting ATPase ATP-binding subunit / ATP phosphohydrolase [potassium-transporting] B chain / Potassium-binding and translocating subunit ...ATP phosphohydrolase [potassium-transporting] B chain / Potassium-binding and translocating subunit B / Potassium-translocating ATPase B chain


Mass: 72251.867 Da / Num. of mol.: 1 / Mutation: S162A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: kdpB, ACU57_12810, AUQ13_21365, BANRA_01741, BANRA_03871, BJJ90_18860, BMA87_25490, BUE81_20115, BvCms12BK_01600, BvCms2454_04068, BvCmsHHP001_00773, BvCmsKSP026_01798, BW690_07915, C5F73_ ...Gene: kdpB, ACU57_12810, AUQ13_21365, BANRA_01741, BANRA_03871, BJJ90_18860, BMA87_25490, BUE81_20115, BvCms12BK_01600, BvCms2454_04068, BvCmsHHP001_00773, BvCmsKSP026_01798, BW690_07915, C5F73_11290, C5N07_14655, C9E25_26860, CA593_25670, CG692_12950, D0X26_17505, D2185_15880, D3821_21860, D3Y67_05225, D9C99_19310, D9D44_15885, D9G69_25000, D9J52_12830, D9Z28_23020, DBQ99_18015, DJ503_06885, DL326_22935, DNC98_20165, DT034_24060, DTL43_19975, DXT73_03325, E2119_26470, E2134_18590, E2135_05105, E4K51_13740, E4K53_14005, E4K55_14010, E4K61_12660, EA213_10640, EAI52_22455, EC3234A_8c00240, ECTO6_03351, ED307_21635, EEP23_23700, EG808_19375, EI021_12885, EI028_12885, EI041_12805, EPT01_08650, ERS085365_04785, ERS085416_02368, ERS139211_04507, ERS150873_01995, EXX71_13725, EYD11_15985, EYV18_15890, F1E19_24885, FNW97_03510, FV293_12590, FWK02_15235, GKF39_08175, GKF74_06655, GKF86_08100, GKF89_05800, GP689_15940, GQE33_23975, GQE34_25255, GQE64_10535, GQM17_23760, GRW05_21645, GRW42_11030, GRW80_11130, GRW81_16515, HVY93_16485, HX136_18170, NCTC8500_03901, NCTC9045_04049, NCTC9062_04760, PGD_02634, RK56_025170, SAMEA3472047_02084, SAMEA3484427_03079, SAMEA3484429_03303, SAMEA3753300_04145, WP2S18E08_32500, WP7S17E04_30590
Production host: Escherichia coli (E. coli) / Strain (production host): LB2003 / References: UniProt: A0A024L5I2, P-type K+ transporter

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Non-polymers , 3 types, 11 molecules

#5: Chemical
ChemComp-RB / RUBIDIUM ION / Rubidium


Mass: 85.468 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Rb / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-CDL / CARDIOLIPIN / DIPHOSPHATIDYL GLYCEROL / BIS-(1,2-DIACYL-SN-GLYCERO-3-PHOSPHO)-1',3'-SN-GLYCEROL / Cardiolipin


Mass: 1464.043 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C81H156O17P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#7: Chemical ChemComp-ACP / PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / ADENOSINE-5'-[BETA, GAMMA-METHYLENE]TRIPHOSPHATE


Mass: 505.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H18N5O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PCP, energy-carrying molecule analogue*YM

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Details

Has ligand of interestY

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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: KdpFABC / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.157 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: LB2003
Buffer solutionpH: 8
Details: 10 mM Tris-HCl pH 8, 10 mM MgCl2, 10 mM NaCl and 0.0125% DDM
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49407 X / Calibrated magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 8 / Num. of real images: 22046
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEM3.9betaimage acquisition
4CTFFIND4.1.13CTF correction
7Cootmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 756834
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196682 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 6HRA

6hra

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00411288
ELECTRON MICROSCOPYf_angle_d0.76215320
ELECTRON MICROSCOPYf_dihedral_angle_d9.3281683
ELECTRON MICROSCOPYf_chiral_restr0.0461823
ELECTRON MICROSCOPYf_plane_restr0.0051919

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