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- PDB-6vxo: NaChBac-Nav1.7VSDII chimera in nanodisc -

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Basic information

Entry
Database: PDB / ID: 6vxo
TitleNaChBac-Nav1.7VSDII chimera in nanodisc
ComponentsNaChBac-Nav1.7VSDII chimera
KeywordsMEMBRANE PROTEIN / NaChBac / Channels / Sodium Ion-Selective
Function / homology
Function and homology information


action potential propagation / detection of mechanical stimulus involved in sensory perception / cardiac muscle cell action potential involved in contraction / node of Ranvier / voltage-gated sodium channel complex / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / behavioral response to pain ...action potential propagation / detection of mechanical stimulus involved in sensory perception / cardiac muscle cell action potential involved in contraction / node of Ranvier / voltage-gated sodium channel complex / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / behavioral response to pain / neuronal action potential / axon terminus / sensory perception of pain / sodium ion transmembrane transport / post-embryonic development / response to toxic substance / circadian rhythm / Sensory perception of sweet, bitter, and umami (glutamate) taste / inflammatory response / axon / plasma membrane
Similarity search - Function
Voltage-gated potassium channels. Chain C / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Helix Hairpins - #70 / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...Voltage-gated potassium channels. Chain C / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Helix Hairpins - #70 / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Voltage-dependent channel domain superfamily / Four Helix Bundle (Hemerythrin (Met), subunit A) / Ion transport domain / Ion transport protein / Helix Hairpins / Up-down Bundle / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-POV / Sodium channel protein type 9 subunit alpha / BH1501 protein
Similarity search - Component
Biological speciesBacillus halodurans C-125 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYan, N. / Gao, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1420541 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na channels in nanodisc.
Authors: Shuai Gao / William C Valinsky / Nguyen Cam On / Patrick R Houlihan / Qian Qu / Lei Liu / Xiaojing Pan / David E Clapham / Nieng Yan /
Abstract: NaChBac, the first bacterial voltage-gated Na (Na) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Na channels. Discovered nearly ...NaChBac, the first bacterial voltage-gated Na (Na) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Na channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NaAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Na channels in lipid bilayers. To study GMTs in mammalian Na channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Na1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Na1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Na channels in a membrane environment.
History
DepositionFeb 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 8, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: NaChBac-Nav1.7VSDII chimera
C: NaChBac-Nav1.7VSDII chimera
D: NaChBac-Nav1.7VSDII chimera
E: NaChBac-Nav1.7VSDII chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,72816
Polymers127,6074
Non-polymers9,12112
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
NaChBac-Nav1.7VSDII chimera / Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / Sodium channel protein ...Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / Sodium channel protein type IX subunit alpha / Voltage-gated sodium channel subunit alpha Nav1.7


Mass: 31901.695 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus halodurans C-125 (bacteria), (gene. exp.) Homo sapiens (human)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Gene: BH1501, SCN9A, NENA / Production host: Escherichia coli (E. coli) / References: UniProt: Q9KCR8, UniProt: Q15858
#2: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
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: NaChBac-Nav1.7VSDII chimera / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisC4H11NO31
2150 mMsodium chlorideNaCl1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: NaChBac-Nav1.7VSDII chimera in lipid nanodisc
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: blot for 5 seconds before plunging

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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
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17rc2_3619: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31147 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0088244
ELECTRON MICROSCOPYf_angle_d0.76511112
ELECTRON MICROSCOPYf_dihedral_angle_d17.5724828
ELECTRON MICROSCOPYf_chiral_restr0.0471260
ELECTRON MICROSCOPYf_plane_restr0.0051272

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