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- EMDB-21560: NaChBac-Nav1.7VSDII chimera and HWTX-IV complex -

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Basic information

Entry
Database: EMDB / ID: EMD-21560
TitleNaChBac-Nav1.7VSDII chimera and HWTX-IV complex
Map dataNaChBac-Nav1.7VSDII chimera and HWTX-IV complex
Sample
  • Complex: NaChBac-Nav1.7VSDII chimera - HWTX-IV complex
    • Complex: NaChBac-Nav1.7VSDII chimera
      • Protein or peptide: NaChBac-Nav1.7VSDII chimera
    • Complex: HWTX-IV
      • Protein or peptide: Huwentoxin-IV
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / host cell presynaptic membrane / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / ion channel inhibitor activity / sodium ion transport / behavioral response to pain / voltage-gated calcium channel complex ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / host cell presynaptic membrane / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / ion channel inhibitor activity / sodium ion transport / behavioral response to pain / voltage-gated calcium channel complex / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / calcium ion import across plasma membrane / sodium ion transmembrane transport / sodium channel regulator activity / monoatomic cation channel activity / sensory perception of pain / post-embryonic development / Sensory perception of sweet, bitter, and umami (glutamate) taste / response to toxic substance / circadian rhythm / toxin activity / inflammatory response / axon / extracellular region / plasma membrane
Similarity search - Function
Huwentoxin-1 family signature. / Huwentoxin, conserved site-1 / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit ...Huwentoxin-1 family signature. / Huwentoxin, conserved site-1 / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Huwentoxin-IV / Sodium channel protein type 9 subunit alpha / BH1501 protein
Similarity search - Component
Biological speciesBacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) (bacteria) / Cyriopagopus schmidti (Chinese earth tiger)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsYan N / Gao S
Funding support United States, 1 items
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
DepositionMar 17, 2020-
Header (metadata) releaseApr 15, 2020-
Map releaseJun 24, 2020-
UpdateJan 13, 2021-
Current statusJan 13, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6w6o
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21560.png

Downloads & links

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Map

FileDownload / File: emd_21560.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNaChBac-Nav1.7VSDII chimera and HWTX-IV complex
Voxel sizeX=Y=Z: 1.114 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.0790109 - 0.1573281
Average (Standard dev.)-2.6721114e-05 (±0.0034513245)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 267.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1141.1141.114
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z267.360267.360267.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0790.157-0.000

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Supplemental data

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Sample components

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Entire : NaChBac-Nav1.7VSDII chimera - HWTX-IV complex

EntireName: NaChBac-Nav1.7VSDII chimera - HWTX-IV complex
Components
  • Complex: NaChBac-Nav1.7VSDII chimera - HWTX-IV complex
    • Complex: NaChBac-Nav1.7VSDII chimera
      • Protein or peptide: NaChBac-Nav1.7VSDII chimera
    • Complex: HWTX-IV
      • Protein or peptide: Huwentoxin-IV
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate

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Supramolecule #1: NaChBac-Nav1.7VSDII chimera - HWTX-IV complex

SupramoleculeName: NaChBac-Nav1.7VSDII chimera - HWTX-IV complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: NaChBac-Nav1.7VSDII chimera

SupramoleculeName: NaChBac-Nav1.7VSDII chimera / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: HWTX-IV

SupramoleculeName: HWTX-IV / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Cyriopagopus schmidti (Chinese earth tiger)

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Macromolecule #1: NaChBac-Nav1.7VSDII chimera

MacromoleculeName: NaChBac-Nav1.7VSDII chimera / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Molecular weightTheoretical: 31.901695 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKMEARQKQN SFTSKMQKIV NHRAFTFTVI ALILFNALIV GIETYPRIYA DHKWLFYRID LVLLWIFTIE IAMRFLASNP KSAFFRSSW NWFDFLIVTL SLVELFLADV EGLSVLRILR VLRVLRAISV VPSLRRLVDA LVMTIPALGN ILILMSIFFY I FAVIGTML ...String:
MKMEARQKQN SFTSKMQKIV NHRAFTFTVI ALILFNALIV GIETYPRIYA DHKWLFYRID LVLLWIFTIE IAMRFLASNP KSAFFRSSW NWFDFLIVTL SLVELFLADV EGLSVLRILR VLRVLRAISV VPSLRRLVDA LVMTIPALGN ILILMSIFFY I FAVIGTML FQHVSPEYFG NLQLSLLTLF QVVTLESWAS GVMRPIFAEV PWSWLYFVSF VLIGTFIIFN LFIGVIVNNV EK AELTDNE EDGEADGLKQ EISALRKDVA ELKSLLKQSK

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Macromolecule #2: Huwentoxin-IV

MacromoleculeName: Huwentoxin-IV / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Cyriopagopus schmidti (Chinese earth tiger)
Molecular weightTheoretical: 4.120865 KDa
SequenceString:
ECLEIFKACN PSNDQCCKSS KLVCSRKTRW CKYQI

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Macromolecule #3: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 3 / Number of copies: 12 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM / POPC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 10
Component:
ConcentrationFormulaName
25.0 mMC2H5NO2Glycine
150.0 mMNaClSodium chloridesodium chloride
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted for 5 seconds before plunging.
DetailsNaChBac was in lipid nanodisc.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 49149

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