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- PDB-6w6o: NaChBac-Nav1.7VSDII chimera and HWTX-IV complex -

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Basic information

Entry
Database: PDB / ID: 6w6o
TitleNaChBac-Nav1.7VSDII chimera and HWTX-IV complex
Components
  • Huwentoxin-IV
  • NaChBac-Nav1.7VSDII chimera
KeywordsTRANSPORT PROTEIN/TOXIN / NaChBac / Channels / Sodium Ion-Selective / TRANSPORT PROTEIN-TOXIN complex
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 / host cell presynaptic membrane / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / ion channel inhibitor activity / Phase 0 - rapid depolarisation ...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 / host cell presynaptic membrane / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / ion channel inhibitor activity / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / behavioral response to pain / sodium channel regulator activity / neuronal action potential / axon terminus / sensory perception of pain / sodium ion transmembrane transport / post-embryonic development / circadian rhythm / response to toxic substance / Sensory perception of sweet, bitter, and umami (glutamate) taste / toxin activity / inflammatory response / axon / extracellular region / plasma membrane
Similarity search - Function
Huwentoxin, conserved site-1 / Huwentoxin-1 family signature. / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated potassium channels. Chain C / 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 ...Huwentoxin, conserved site-1 / Huwentoxin-1 family signature. / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated potassium channels. Chain C / 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 / SCN5A-like, C-terminal IQ motif / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Helix Hairpins - #70 / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Voltage-dependent channel domain superfamily / Four Helix Bundle (Hemerythrin (Met), subunit A) / Ion transport domain / Ion transport protein / Helix Hairpins / Up-down Bundle / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-POV / Huwentoxin-IV / Sodium channel protein type 9 subunit alpha / BH1501 protein
Similarity search - Component
Biological speciesBacillus halodurans (bacteria)
Homo sapiens (human)
Cyriopagopus schmidti (Chinese earth tiger)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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.
History
DepositionMar 17, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: NaChBac-Nav1.7VSDII chimera
C: Huwentoxin-IV
D: NaChBac-Nav1.7VSDII chimera
E: Huwentoxin-IV
F: NaChBac-Nav1.7VSDII chimera
G: Huwentoxin-IV
H: NaChBac-Nav1.7VSDII chimera
I: Huwentoxin-IV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,21120
Polymers144,0908
Non-polymers9,12112
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22840 Å2
ΔGint-175 kcal/mol
Surface area55220 Å2

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Components

#1: Protein
NaChBac-Nav1.7VSDII chimera / BH1501 protein / Sodium channel protein type 9 subunit alpha / BH1501 protein


Mass: 31901.695 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) (bacteria), (gene. exp.) Homo sapiens (human)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Gene: BH1501, SCN9A, NENA / Production host: Escherichia coli (E. coli) / References: UniProt: Q9KCR8, UniProt: Q15858
#2: Protein/peptide
Huwentoxin-IV / HwTx-IV / Huwentoxin-4 / Huwentoxin-IVa / HWTX-IVa / Huwentoxin-IVb / HWTX-IVb / Huwentoxin-IVc / ...HwTx-IV / Huwentoxin-4 / Huwentoxin-IVa / HWTX-IVa / Huwentoxin-IVb / HWTX-IVb / Huwentoxin-IVc / HWTX-IVc / Mu-theraphotoxin-Hs2a / Mu-TRTX-Hs2a


Mass: 4120.865 Da / Num. of mol.: 4 / Fragment: UNP residues 53-87 / Source method: obtained synthetically
Source: (synth.) Cyriopagopus schmidti (Chinese earth tiger)
References: UniProt: P83303
#3: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
Has ligand of interestN
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1NaChBac-Nav1.7VSDII chimera - HWTX-IV complexCOMPLEX#1-#20MULTIPLE SOURCES
2NaChBac-Nav1.7VSDII chimeraCOMPLEX#11RECOMBINANT
3HWTX-IVCOMPLEX#21NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
12Bacillus halodurans (strain ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125) (bacteria)272558ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
23Cyriopagopus schmidti (Chinese earth tiger)29017
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 10
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMGlycineC2H5NO21
2150 mMsodium chlorideNaCl1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: NaChBac was in lipid nanodisc.
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: Blotted 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 FIELD
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: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49149 / Symmetry type: POINT

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