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- PDB-6p6x: Crystal structure of voltage-gated sodium channel NavAb G94C/Q150... -

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Basic information

Entry
Database: PDB / ID: 6p6x
TitleCrystal structure of voltage-gated sodium channel NavAb G94C/Q150C mutant in the activated state
ComponentsIon transport proteinIon transporter
Keywordsmembrane protein / metal transport / Ion channel / ion transport protein
Function / homologyVoltage-gated cation channel calcium and sodium / Voltage-dependent channel domain superfamily / monoatomic cation channel activity / Ion transport domain / Ion transport protein / identical protein binding / plasma membrane / 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Ion transport protein
Function and homology information
Biological speciesArcobacter butzleri (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.75 Å
AuthorsWisedchaisri, G. / Tonggu, L. / McCord, E. / Gamal El-Din, T.M. / Wang, L. / Zheng, N. / Catterall, W.A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS015751 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL112808 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM007750 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell / Year: 2019
Title: Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel.
Authors: Goragot Wisedchaisri / Lige Tonggu / Eedann McCord / Tamer M Gamal El-Din / Liguo Wang / Ning Zheng / William A Catterall /
Abstract: Voltage-gated sodium (Na) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state ...Voltage-gated sodium (Na) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.
History
DepositionJun 4, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Nov 20, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ion transport protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)37,15112
Polymers29,8101
Non-polymers7,34011
Water0
1
A: Ion transport protein
hetero molecules

A: Ion transport protein
hetero molecules

A: Ion transport protein
hetero molecules

A: Ion transport protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,60248
Polymers119,2424
Non-polymers29,36144
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_445-x-1,-y-1,z1
crystal symmetry operation3_455-y-1,x,z1
crystal symmetry operation4_545y,-x-1,z1
Buried area50300 Å2
ΔGint-320 kcal/mol
Surface area39240 Å2
MethodPISA
Unit cell
Length a, b, c (Å)124.449, 124.449, 193.339
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number97
Space group name H-MI422

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Components

#1: Protein Ion transport protein / Ion transporter


Mass: 29810.398 Da / Num. of mol.: 1 / Mutation: G94C, Q150C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arcobacter butzleri (strain RM4018) (bacteria)
Strain: RM4018 / Gene: Abu_1752 / Variant: RM4018 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A8EVM5
#2: Chemical
ChemComp-PX4 / 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Dimyristoylphosphatidylcholine


Mass: 678.940 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C36H73NO8P / Comment: DMPC, phospholipid*YM
#3: Chemical ChemComp-CPS / 3-[(3-CHOLAMIDOPROPYL)DIMETHYLAMMONIO]-1-PROPANESULFONATE / CHAPS / CHAPS detergent


Mass: 614.877 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C32H58N2O7S / Comment: detergent*YM
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 6.28 Å3/Da / Density % sol: 80.41 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 5.6 / Details: 1.8 M ammonium sulfate 0.1 M sodium citrate pH 5.6

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Sep 7, 2018
RadiationMonochromator: Double-crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.75→50 Å / Num. obs: 19146 / % possible obs: 94.8 % / Redundancy: 19.6 % / Biso Wilson estimate: 26.5 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.117 / Rpim(I) all: 0.026 / Rrim(I) all: 0.12 / Χ2: 0.962 / Net I/σ(I): 27.75
Reflection shellResolution: 2.75→2.8 Å / Redundancy: 8.4 % / Mean I/σ(I) obs: 1 / Num. unique obs: 556 / CC1/2: 0.63 / Rpim(I) all: 0.375 / Χ2: 0.878 / % possible all: 56.3

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
Cootmodel building
REFMAC5.8.0238refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3RVY

3rvy


Resolution: 2.75→44.04 Å / Cor.coef. Fo:Fc: 0.908 / Cor.coef. Fo:Fc free: 0.861 / SU B: 8.8 / SU ML: 0.18 / Cross valid method: THROUGHOUT / ESU R: 0.347 / ESU R Free: 0.292 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.26543 851 5.1 %RANDOM
Rwork0.21321 ---
obs0.21579 15906 83.24 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 40.734 Å2
Baniso -1Baniso -2Baniso -3
1-0.1 Å20 Å20 Å2
2--0.1 Å20 Å2
3----0.19 Å2
Refinement stepCycle: 1 / Resolution: 2.75→44.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1817 0 342 0 2159
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0132213
X-RAY DIFFRACTIONr_bond_other_d0.0010.0172309
X-RAY DIFFRACTIONr_angle_refined_deg1.7411.6632972
X-RAY DIFFRACTIONr_angle_other_deg1.1821.5415338
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.8845228
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.142078
X-RAY DIFFRACTIONr_dihedral_angle_3_deg20.7115307
X-RAY DIFFRACTIONr_dihedral_angle_4_deg20.1251510
X-RAY DIFFRACTIONr_chiral_restr0.0770.2302
X-RAY DIFFRACTIONr_gen_planes_refined0.0080.022080
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02450
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.7983.754912
X-RAY DIFFRACTIONr_mcbond_other3.7883.749911
X-RAY DIFFRACTIONr_mcangle_it5.8625.6131137
X-RAY DIFFRACTIONr_mcangle_other5.865.621138
X-RAY DIFFRACTIONr_scbond_it4.5974.921300
X-RAY DIFFRACTIONr_scbond_other4.5964.9231301
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other7.347.171835
X-RAY DIFFRACTIONr_long_range_B_refined9.47848.2062380
X-RAY DIFFRACTIONr_long_range_B_other9.47648.2372381
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.751→2.823 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.353 9 -
Rwork0.269 208 -
obs--14.83 %

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