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- EMDB-24580: Human ASIC1a-Nb.C1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-24580
TitleHuman ASIC1a-Nb.C1 complex
Map dataASIC1a-Nb.C1 complex
Sample
  • Complex: Human ASIC1a in complex with Nb.C1
    • Protein or peptide: Nanobodies Nb.C1
    • Protein or peptide: Acid-sensing ion channel 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 speciesHomo sapiens (human) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsWu Y / Chen Z / Sigworth FJ / Canessa CM
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00995
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.00995
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7rnn
  • Surface level: 0.00995
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7rnn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24580.png

Downloads & links

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Map

FileDownload / File: emd_24580.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationASIC1a-Nb.C1 complex
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.00995 / Movie #1: 0.00995
Minimum - Maximum-0.0207817 - 0.048160475
Average (Standard dev.)0.00013074193 (±0.00136802)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z249.000249.000249.000
α/β/γ90.00090.00090.000
start NX/NY/NZ594743
NX/NY/NZ375263
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0210.0480.000

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Supplemental data

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Sample components

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Entire : Human ASIC1a in complex with Nb.C1

EntireName: Human ASIC1a in complex with Nb.C1
Components
  • Complex: Human ASIC1a in complex with Nb.C1
    • Protein or peptide: Nanobodies Nb.C1
    • Protein or peptide: Acid-sensing ion channel 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Human ASIC1a in complex with Nb.C1

SupramoleculeName: Human ASIC1a in complex with Nb.C1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Nanobodies Nb.C1

MacromoleculeName: Nanobodies Nb.C1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 12.76221 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQPRGSLRL SCAASGFTFS RAAMSWYRQA PGKEREMVST IGSFGVSTNY SDSVKGRFTI SRDNAKNTVY LHMNSLKPE DTAVYYCNAR YRSSYPWGQG TQVTVSS

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Macromolecule #2: Acid-sensing ion channel 1

MacromoleculeName: Acid-sensing ion channel 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.001309 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MELKAEEEEV GGVQPVSIQA FASSSTLHGL AHIFSYERLS LKRALWALCF LGSLAVLLCV CTERVQYYFH YHHVTKLDEV AASQLTFPA VTFCNLNEFR FSQVSKNDLY HAGELLALLN NRYEIPDTQM ADEKQLEILQ DKANFRSFKP KPFNMREFYD R AGHDIRDM ...String:
MELKAEEEEV GGVQPVSIQA FASSSTLHGL AHIFSYERLS LKRALWALCF LGSLAVLLCV CTERVQYYFH YHHVTKLDEV AASQLTFPA VTFCNLNEFR FSQVSKNDLY HAGELLALLN NRYEIPDTQM ADEKQLEILQ DKANFRSFKP KPFNMREFYD R AGHDIRDM LLSCHFRGEV CSAEDFKVVF TRYGKCYTFN SGRDGRPRLK TMKGGTGNGL EIMLDIQQDE YLPVWGETDE TS FEAGIKV QIHSQDEPPF IDQLGFGVAP GFQTFVACQE QRLIYLPPPW GTCKAVTMDS DLDFFDSYSI TACRIDCETR YLV ENCNCR MVHMPGDAPY CTPEQYKECA DPALDFLVEK DQEYCVCEMP CNLTRYGKEL SMVKIPSKAS AKYLAKKYNK SEQY IGENI LVLDIFFEVL NYETIEQKKA YEIAGLLGDI GGQMGLFIGA SILTVLELFD YAYEVIKHKL CRRGKCQKEA KRSSA DKGV ALSLDDVKRH NPCEGLRGHP AGMTYAANIL PHHPARGTFE DFTC

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration3.8 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium Chloride
5.0 mMCaCl2Calcium Chloride
0.5 mg/mLC24H46O11DDM

Details: Solutions were filtered with 0.22um to avoid contamination.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK III / Details: blot for 3s before plunging.
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPreliminary grid screening was performed manually
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 7318 / Average exposure time: 1.4 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1287029 / Details: Template (emd_7009) based particle picking.
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 4 / Avg.num./class: 84500 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 84500
DetailsThe selected images were high-pass filtered and normalized.

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Atomic model buiding 1

Initial model
PDB IDChain

6vtl

chain_id: A

5ivo

chain_id: A
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 200 / Target criteria: Correlation coefficient
Output model

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

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