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- EMDB-21428: NaChBac in GDN -

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Basic information

Entry
Database: EMDB / ID: EMD-21428
TitleNaChBac in GDN
Map data
Sample
  • Complex: NaChBac
    • Protein or peptide: BH1501 protein
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
Function / homologyVoltage-gated cation channel calcium and sodium / Voltage-dependent channel domain superfamily / monoatomic cation channel activity / Ion transport domain / Ion transport protein / plasma membrane / BH1501 protein
Function and homology information
Biological speciesBacillus halodurans C-125 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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 21, 2020-
Header (metadata) releaseJun 24, 2020-
Map releaseJun 24, 2020-
UpdateJul 8, 2020-
Current statusJul 8, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vx3
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21428.png

Downloads & links

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Map

FileDownload / File: emd_21428.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: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.13355376 - 0.20315522
Average (Standard dev.)0.00026458732 (±0.00431609)
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-0.1340.2030.000

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Supplemental data

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Sample components

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Entire : NaChBac

EntireName: NaChBac
Components
  • Complex: NaChBac
    • Protein or peptide: BH1501 protein
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate

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Supramolecule #1: NaChBac

SupramoleculeName: NaChBac / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: BH1501 protein

MacromoleculeName: BH1501 protein / 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: 31.486197 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKMEARQKQN SFTSKMQKIV NHRAFTFTVI ALILFNALIV GIETYPRIYA DHKWLFYRID LVLLWIFTIE IAMRFLASNP KSAFFRSSW NWFDFLIVAA GHIFAGAQFV TVLRILRVLR VLRAISVVPS LRRLVDALVM TIPALGNILI LMSIFFYIFA V IGTMLFQH ...String:
MKMEARQKQN SFTSKMQKIV NHRAFTFTVI ALILFNALIV GIETYPRIYA DHKWLFYRID LVLLWIFTIE IAMRFLASNP KSAFFRSSW NWFDFLIVAA GHIFAGAQFV TVLRILRVLR VLRAISVVPS LRRLVDALVM TIPALGNILI LMSIFFYIFA V IGTMLFQH VSPEYFGNLQ LSLLTLFQVV TLESWASGVM RPIFAEVPWS WLYFVSFVLI GTFIIFNLFI GVIVNNVEKA EL TDNEEDG EADGLKQEIS ALRKDVAELK SLLKQSK

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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: 8 / 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: 10
Component:
ConcentrationFormulaName
25.0 mMC2H5NO2Glycine
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 was 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

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91616
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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