|Entry||Database: PDB / ID: 6vwx|
|Title||NaChBac in lipid nanodisc|
|Keywords||MEMBRANE PROTEIN / NaChBac / Channels / Sodium Ion-Selective|
|Function / homology|
Function and homology information
cation channel activity / integral component of membrane / plasma membrane
Ion transport domain / Voltage-dependent channel domain superfamily / Voltage-gated cation channel calcium and sodium / Helix Hairpins - #70 / Helix Hairpins / Orthogonal Bundle / Mainly Alpha
|Biological species||Bacillus halodurans C-125 (bacteria)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å|
|Authors||Yan, N. / Gao, S.|
|Funding support|| United States, 1items |
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
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A: BH1501 protein
D: BH1501 protein
E: BH1501 protein
F: BH1501 protein
Mass: 31486.197 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus halodurans C-125 (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Gene: BH1501 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9KCR8
ChemComp-POV / (
Mass: 760.076 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C42H82NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
|Has ligand of interest||Y|
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: NaChBac / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT|
|Source (natural)||Organism: Bacillus halodurans C-125 (bacteria)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 8|
|Specimen||Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: NaChBac was in lipid nanodisc|
|Specimen support||Details: no / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: blot for 5 seconds before plunging|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 53 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|3D reconstruction||Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 281039 / Symmetry type: POINT|
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