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- PDB-8jfz: Cryo-EM structure of Na+,K+-ATPase in the E1.Mg2+ state. -

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Basic information

Entry
Database: PDB / ID: 8jfz
TitleCryo-EM structure of Na+,K+-ATPase in the E1.Mg2+ state.
Components
  • FXYD domain-containing ion transport regulator
  • Na+,K+-ATPase beta subunit
  • Na, K-ATPase alpha subunit
KeywordsMEMBRANE PROTEIN / ion pump / P-type ATPase
Function / homology
Function and homology information


regulation of monoatomic ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / sodium channel regulator activity / monoatomic ion transport ...regulation of monoatomic ion transport / P-type sodium:potassium-exchanging transporter activity / sodium:potassium-exchanging ATPase complex / sodium ion export across plasma membrane / intracellular potassium ion homeostasis / intracellular sodium ion homeostasis / potassium ion import across plasma membrane / ATPase activator activity / sodium channel regulator activity / monoatomic ion transport / proton transmembrane transport / ATP hydrolysis activity / ATP binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
Ion-transport regulator, FXYD motif / : / ATP1G1/PLM/MAT8 family / FXYD family signature. / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / : / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / Sodium and potassium ATPases beta subunits signature 2. ...Ion-transport regulator, FXYD motif / : / ATP1G1/PLM/MAT8 family / FXYD family signature. / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit beta superfamily / : / Sodium / potassium ATPase beta chain / Sodium and potassium ATPases beta subunits signature 1. / Sodium and potassium ATPases beta subunits signature 2. / P-type ATPase subfamily IIC, subunit alpha / 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
CHOLESTEROL / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Sodium/potassium-transporting ATPase subunit beta / Sodium/potassium-transporting ATPase subunit alpha / FXYD domain-containing ion transport regulator
Similarity search - Component
Biological speciesSqualus acanthias (spiny dogfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKanai, R. / Vilsen, B. / Cornelius, F. / Toyoshima, C.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP19H00975 Japan
Japan Society for the Promotion of Science (JSPS)JP21K06109 Japan
CitationJournal: FEBS Lett / Year: 2023
Title: Crystal structures of Na ,K -ATPase reveal the mechanism that converts the K -bound form to Na -bound form and opens and closes the cytoplasmic gate.
Authors: Ryuta Kanai / Bente Vilsen / Flemming Cornelius / Chikashi Toyoshima /
Abstract: Na ,K -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na and K across the cell membrane by alternating between the E1 (showing high affinity for Na and low affinity ...Na ,K -ATPase (NKA) plays a pivotal role in establishing electrochemical gradients for Na and K across the cell membrane by alternating between the E1 (showing high affinity for Na and low affinity for K ) and E2 (low affinity to Na and high affinity to K ) forms. Presented here are two crystal structures of NKA in E1·Mg and E1·3Na states at 2.9 and 2.8 Å resolution, respectively. These two E1 structures fill a gap in our description of the NKA reaction cycle based on the atomic structures. We describe how NKA converts the K -bound E2·2K form to an E1 (E1·Mg ) form, which allows high-affinity Na binding, eventually closing the cytoplasmic gate (in E1 ~ P·ADP·3Na ) after binding three Na , while keeping the extracellular ion pathway sealed. We now understand previously unknown functional roles for several parts of NKA and that NKA uses even the lipid bilayer for gating the ion pathway.
History
DepositionMay 19, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Na+,K+-ATPase beta subunit
C: Na, K-ATPase alpha subunit
E: FXYD domain-containing ion transport regulator
B: Na+,K+-ATPase beta subunit
A: Na, K-ATPase alpha subunit
G: FXYD domain-containing ion transport regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)343,27746
Polymers317,3646
Non-polymers25,91340
Water1086
1
D: Na+,K+-ATPase beta subunit
C: Na, K-ATPase alpha subunit
E: FXYD domain-containing ion transport regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,25222
Polymers158,6823
Non-polymers12,57019
Water181
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: Na+,K+-ATPase beta subunit
A: Na, K-ATPase alpha subunit
G: FXYD domain-containing ion transport regulator
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,02524
Polymers158,6823
Non-polymers13,34321
Water181
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A" or chain "B" or chain "G"
d_2ens_1chain "C" or chain "D" or chain "E"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11GLYGLYSERSERBD12 - 30512 - 305
d_12NAGNAGNAGNAGKK1
d_13NAGNAGNAGNAGKK2
d_14FUCFUCFUCFUCKK6
d_15BMABMABMABMAKK3
d_16MANMANMANMANKK4
d_17MANMANMANMANKK5
d_18NAGNAGNAGNAGLL1
d_19NAGNAGNAGNAGLL2
d_110BMABMABMABMALL3
d_111MANMANMANMANLL6
d_112MANMANMANMANLL4
d_113NAGNAGNAGNAGLL5
d_114NAGNAGNAGNAGMM1
d_115NAGNAGNAGNAGMM2
d_116FUCFUCFUCFUCMM5
d_117BMABMABMABMAMM3
d_118MANMANMANMANMM4
d_119NAGNAGNAGNAGNN1
d_120NAGNAGNAGNAGNN2
d_121CLRCLRCLRCLRAFA1102
d_122CLRCLRCLRCLRBDA401
d_123LYSLYSTYRTYRAE37 - 102342 - 1028
d_124CLRCLRCLRCLRGSA1301
d_125CLRCLRCLRCLRAGA1103
d_126PCWPCWPCWPCWAHA1104
d_127PCWPCWPCWPCWAIA1105
d_128PCWPCWPCWPCWAJA1106
d_129PCWPCWPCWPCWAKA1107
d_130PCWPCWPCWPCWALA1108
d_131PCWPCWPCWPCWAMA1109
d_132PCWPCWPCWPCWGTA1302
d_133PCWPCWPCWPCWANA1110
d_134PCWPCWPCWPCWAOA1111
d_135PCWPCWPCWPCWAPA1112
d_136MGMGMGMGAQA1113
d_137MGMGMGMGARA1114
d_138GLUGLUCYSCYSGF4 - 4324 - 63
d_21GLYGLYSERSERDA12 - 30512 - 305
d_22NAGNAGNAGNAGFG1
d_23NAGNAGNAGNAGFG2
d_24FUCFUCFUCFUCFG6
d_25BMABMABMABMAFG3
d_26MANMANMANMANFG4
d_27MANMANMANMANFG5
d_28NAGNAGNAGNAGHH1
d_29NAGNAGNAGNAGHH2
d_210BMABMABMABMAHH3
d_211MANMANMANMANHH6
d_212MANMANMANMANHH4
d_213NAGNAGNAGNAGHH5
d_214NAGNAGNAGNAGII1
d_215NAGNAGNAGNAGII2
d_216FUCFUCFUCFUCII5
d_217BMABMABMABMAII3
d_218MANMANMANMANII4
d_219NAGNAGNAGNAGJJ1
d_220NAGNAGNAGNAGJJ2
d_221CLRCLRCLRCLRCP1101
d_222CLRCLRCLRCLRDO401
d_223LYSLYSTYRTYRCB37 - 102342 - 1028
d_224CLRCLRCLRCLREBA1301
d_225CLRCLRCLRCLRAEA1101
d_226PCWPCWPCWPCWCQ1102
d_227PCWPCWPCWPCWCR1103
d_228PCWPCWPCWPCWCS1104
d_229PCWPCWPCWPCWCT1105
d_230PCWPCWPCWPCWCU1106
d_231PCWPCWPCWPCWCV1107
d_232PCWPCWPCWPCWECA1302
d_233PCWPCWPCWPCWCW1108
d_234PCWPCWPCWPCWCX1109
d_235PCWPCWPCWPCWCY1110
d_236MGMGMGMGCZ1111
d_237MGMGMGMGCAA1112
d_238GLUGLUCYSCYSEC4 - 4324 - 63

NCS oper: (Code: givenMatrix: (-0.999832324478, -0.00546802466492, 0.0174763736471), (0.00561602233723, -0.999948691204, 0.00843061401413), (0.01742937815, 0.0085273481113, 0.999811732833)Vector: 256. ...NCS oper: (Code: given
Matrix: (-0.999832324478, -0.00546802466492, 0.0174763736471), (0.00561602233723, -0.999948691204, 0.00843061401413), (0.01742937815, 0.0085273481113, 0.999811732833)
Vector: 256.710347816, 256.49918092, -2.90799876279)

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Components

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Protein , 3 types, 6 molecules DBCAEG

#1: Protein Na+,K+-ATPase beta subunit / Sodium/potassium-transporting ATPase subunit beta-1


Mass: 35176.125 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Squalus acanthias (spiny dogfish) / References: UniProt: C4IX13
#2: Protein Na, K-ATPase alpha subunit


Mass: 113309.891 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Squalus acanthias (spiny dogfish) / References: UniProt: Q4H132
#3: Protein FXYD domain-containing ion transport regulator


Mass: 10195.847 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Squalus acanthias (spiny dogfish) / References: UniProt: Q70Q12

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Sugars , 4 types, 8 molecules

#4: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1056.964 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/4,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-4/a4-b1_a6-f1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1114.016 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-1-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][b-D-GlcpNAc]{}}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 894.823 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/4,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-4/a4-b1_a6-e1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#7: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 38 molecules

#8: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: C27H46O
#9: Chemical
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

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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: Na+,K+-ATPase / Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Squalus acanthias (spiny dogfish)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
14 mMmagnesium chlorideMgCl21
230 mMimidazoleC3H4N21
30.01 %C12E8C28H58O91
41 mMDTTC4H10O2S21
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 99.9 % / Chamber temperature: 279 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1RELION3.1.3particle selection
2EPU2.12.0image acquisition
4CTFFIND4.1.14CTF correction
9RELION3.1.3initial Euler assignment
10RELION3.1.3final Euler assignment
12RELION3.1.33D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36635 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8JBM

8jbm


Accession code: 8JBM / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 68.22 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006522770
ELECTRON MICROSCOPYf_angle_d1.335730728
ELECTRON MICROSCOPYf_chiral_restr0.23743540
ELECTRON MICROSCOPYf_plane_restr0.01056420
ELECTRON MICROSCOPYf_dihedral_angle_d16.56629052
Refine LS restraints NCSType: NCS constraints / Rms dev position: 0.00070627441068 Å

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