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- PDB-6vx3: NaChBac in GDN -

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Basic information

Entry
Database: PDB / ID: 6vx3
TitleNaChBac in GDN
ComponentsBH1501 protein
KeywordsMEMBRANE 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
BH1501 protein
Biological speciesBacillus halodurans C-125 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionFeb 21, 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

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Structure visualization

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Assembly

Deposited unit
C: BH1501 protein
A: BH1501 protein
B: BH1501 protein
D: BH1501 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,02512
Polymers125,9454
Non-polymers6,0818
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
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
#2: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 8 / 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 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Bacillus halodurans C-125 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 10
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMGlycineC2H5NO21
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: NaChBac was 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 FIELDBright-field microscopy
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91616 / Symmetry type: POINT

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