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- PDB-7rnn: Human ASIC1a-Nb.C1 complex -

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Basic information

Entry
Database: PDB / ID: 7rnn
TitleHuman ASIC1a-Nb.C1 complex
Components
  • Acid-sensing ion channel 1
  • Nanobodies Nb.C1
KeywordsMEMBRANE PROTEIN / Channel Proton-gated Nanobody
Function / homology
Function and homology information


monoatomic ion-gated channel activity / sensory perception of sour taste / pH-gated monoatomic ion channel activity / ligand-gated sodium channel activity / cellular response to pH / negative regulation of neurotransmitter secretion / neurotransmitter secretion / response to acidic pH / sodium ion transport / protein homotrimerization ...monoatomic ion-gated channel activity / sensory perception of sour taste / pH-gated monoatomic ion channel activity / ligand-gated sodium channel activity / cellular response to pH / negative regulation of neurotransmitter secretion / neurotransmitter secretion / response to acidic pH / sodium ion transport / protein homotrimerization / associative learning / sodium ion transmembrane transport / behavioral fear response / response to amphetamine / regulation of membrane potential / calcium ion transmembrane transport / Stimuli-sensing channels / memory / presynapse / Golgi apparatus / cell surface / plasma membrane
Similarity search - Function
Epithelial sodium channel, chordates / Epithelial sodium channel, conserved site / Amiloride-sensitive sodium channels signature. / Epithelial sodium channel / Amiloride-sensitive sodium channel
Similarity search - Domain/homology
Acid-sensing ion channel 1
Similarity search - Component
Biological speciesVicugna pacos (alpaca)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsWu, Y. / Chen, Z. / Sigworth, F.J. / Canessa, C.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R21 MH10107466402 United States
CitationJournal: Elife / Year: 2021
Title: Structure and analysis of nanobody binding to the human ASIC1a ion channel.
Authors: Yangyu Wu / Zhuyuan Chen / Fred J Sigworth / Cecilia M Canessa /
Abstract: ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies ...ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.
History
DepositionJul 29, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 25, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_volume ..._citation.journal_abbrev / _citation.journal_volume / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
C: Nanobodies Nb.C1
D: Acid-sensing ion channel 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)73,2064
Polymers72,7642
Non-polymers4422
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Triangular top view particles
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1630 Å2
ΔGint5 kcal/mol
Surface area27370 Å2

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Components

#1: Antibody Nanobodies Nb.C1


Mass: 12762.210 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pADL-22c / Production host: Escherichia coli (E. coli) / Strain (production host): WK6
#2: Protein Acid-sensing ion channel 1 / / ASIC1 / Amiloride-sensitive cation channel 2 / neuronal / Brain sodium channel 2 / BNaC2


Mass: 60001.309 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293T / Gene: ASIC1, ACCN2, BNAC2 / Plasmid: pcDNA3.1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: P78348
#3: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
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: Human ASIC1a in complex with Nb.C1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Details: Solutions were filtered with 0.22um to avoid contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaClSodium chloride1
35 mMCalcium ChlorideCaCl21
40.5 mg/mLDDMC24H46O111
SpecimenConc.: 3.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: blot for 3s before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.4 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7318

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
7MOLREPmodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.18.2model refinement
14REFMAC5model refinement
Image processingDetails: The selected images were high-pass filtered and normalized.
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1287029 / Details: Template (emd_7009) based particle picking.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84500 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 200 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
16VTL

6vtl

A1
25IVO

5ivo

A1

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