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- EMDB-21446: NaChBac-Nav1.7VSDII chimera in nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-21446
TitleNaChBac-Nav1.7VSDII chimera in nanodisc
Map data
Sample
  • Complex: NaChBac-Nav1.7VSDII chimera
    • Protein or peptide: NaChBac-Nav1.7VSDII chimera
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
KeywordsNaChBac / Channels / Sodium Ion-Selective / MEMBRANE PROTEIN
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 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 / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Voltage-dependent channel domain superfamily ...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 / 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
Sodium channel protein type 9 subunit alpha / BH1501 protein
Similarity search - Component
Biological speciesBacillus halodurans C-125 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 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
DepositionFeb 22, 2020-
Header (metadata) releaseJun 24, 2020-
Map releaseJun 24, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vxo
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21446.png

Downloads & links

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Map

FileDownload / File: emd_21446.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 240 pix.
= 267.36 Å		1.11 Å/pix.
x 240 pix.
= 267.36 Å		1.11 Å/pix.
x 240 pix.
= 267.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.114 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.05584383 - 0.118232206
Average (Standard dev.)0.000033283766 (±0.003268973)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0560.1180.000

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Supplemental data

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Sample components

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Entire : NaChBac-Nav1.7VSDII chimera

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

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Supramolecule #1: NaChBac-Nav1.7VSDII chimera

SupramoleculeName: NaChBac-Nav1.7VSDII chimera / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)

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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 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

UniProtKB: BH1501 protein, Sodium channel protein type 9 subunit alpha, BH1501 protein

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Macromolecule #2: (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: 2 / 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

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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: 8
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3Tris
150.0 mMNaClsodium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 seconds before plunging.
DetailsNaChBac-Nav1.7VSDII chimera in lipid nanodisc

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Electron microscopy

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

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31147
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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