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- PDB-6cpv: MicroED structure of NaK ion channel reveals a process of Na+ par... -

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Basic information

Entry
Database: PDB / ID: 6cpv
TitleMicroED structure of NaK ion channel reveals a process of Na+ partition into the selectivity filter
ComponentsPotassium channel protein
KeywordsTRANSPORT PROTEIN / ion channel / NaK
Function / homology
Function and homology information


potassium channel activity / membrane / identical protein binding / metal ion binding
Similarity search - Function
Two pore domain potassium channel / Helix Hairpins - #70 / Potassium channel domain / Ion channel / Helix Hairpins / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Transporter / Potassium channel protein
Similarity search - Component
Biological speciesBacillus cereus (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.5002 Å
AuthorsLiu, S. / Gonen, T.
Funding support United States, 1items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)1 United States
CitationJournal: Commun Biol / Year: 2018
Title: MicroED structure of the NaK ion channel reveals a Na partition process into the selectivity filter.
Authors: Shian Liu / Tamir Gonen /
Abstract: Sodium (Na) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration ...Sodium (Na) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na are proposed to play critical roles in determining the conductance and the selectivity of Na channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na ion at the entrance of the channel pore. A process of dilation coupled with Na movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.
History
DepositionMar 14, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 12, 2018Provider: repository / Type: Initial release
Revision 1.1Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Oct 4, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_symmetry

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Potassium channel protein
B: Potassium channel protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,80715
Polymers21,4132
Non-polymers39413
Water724
1
A: Potassium channel protein
hetero molecules

A: Potassium channel protein
hetero molecules

A: Potassium channel protein
hetero molecules

A: Potassium channel protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,03540
Polymers42,8264
Non-polymers1,20836
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
Buried area9320 Å2
ΔGint-182 kcal/mol
Surface area17310 Å2
MethodPISA
2
B: Potassium channel protein
hetero molecules

B: Potassium channel protein
hetero molecules

B: Potassium channel protein
hetero molecules

B: Potassium channel protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)43,19420
Polymers42,8264
Non-polymers36816
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,-y,z1
crystal symmetry operation3_555-y,x,z1
crystal symmetry operation4_555y,-x,z1
Buried area7910 Å2
ΔGint-119 kcal/mol
Surface area19700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)68.072, 68.072, 89.300
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number79
Space group name H-MI4
Components on special symmetry positions
IDModelComponents
11A-201-

NA

21A-202-

NA

31A-203-

NA

41A-204-

NA

51A-205-

NA

61A-206-

NA

71A-207-

NA

81A-208-

NA

91B-201-

NA

101B-202-

NA

111B-203-

NA

121B-204-

NA

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Components

#1: Protein Potassium channel protein / / Transporter / Voltage-gated potassium channel


Mass: 10706.538 Da / Num. of mol.: 2 / Fragment: UNP residues 19-110
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus cereus (bacteria)
Gene: A9485_19160, B4155_3291, BACERE00184_02078, CN419_22740, CN950_06075, CN980_22870, COI98_17615, COK18_26145, CON37_12595
Production host: Escherichia coli (E. coli) / References: UniProt: A0A164U772, UniProt: Q81HW2*PLUS
#2: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Na
#3: Chemical ChemComp-MPD / (4S)-2-METHYL-2,4-PENTANEDIOL / 2-Methyl-2,4-pentanediol


Mass: 118.174 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O2 / Comment: precipitant*YM
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: NaK / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Bacillus cereus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.1 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)
EM diffractionCamera length: 1750 mm
EM diffraction shellResolution: 2.5002→3.1486 Å / Fourier space coverage: 0.76 % / Multiplicity: 4.1 / Num. of structure factors: 2685 / Phase residual: 22.68 °
EM diffraction statsFourier space coverage: 81.7 % / High resolution: 2.5002 Å / Num. of intensities measured: 27479 / Num. of structure factors: 5643 / Phase error: 20.27 ° / Phase residual: 20.27 ° / Phase error rejection criteria: 0 / Rmerge: 0.206 / Rsym: 0.206

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Processing

Software
NameVersionClassification
PHENIX(1.12_2829: ???)refinement
Aimless0.5.32data scaling
iMOSFLM7.1.0data reduction
PHASERphasing
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 68.0716 Å / B: 68.0716 Å / C: 89.3 Å / Space group name: I4 / Space group num: 79
CTF correctionType: NONE
3D reconstructionResolution: 2.5002 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 41
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3E89

3e89


Resolution: 2.5→21.992 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.41 / Phase error: 20.26
RfactorNum. reflection% reflection
Rfree0.2632 283 4.89 %
Rwork0.2183 --
obs0.2205 5793 81.68 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0021513
ELECTRON CRYSTALLOGRAPHYf_angle_d0.4122066
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d9.508859
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.036258
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.002249
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5002-3.14860.32511350.26562550ELECTRON CRYSTALLOGRAPHY76
3.1486-21.99250.23781480.20132960ELECTRON CRYSTALLOGRAPHY87

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