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- EMDB-30346: Cryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0 -

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Basic information

Entry
Database: EMDB / ID: EMD-30346
TitleCryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0
Map data
Sample
  • Complex: hASIC1a
    • Protein or peptide: Acid-sensing ion channel 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM IONSodium
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsSun DM / Liu SL / Li SY / Yang F / Tian CL
Funding support China, 8 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31600601 China
National Natural Science Foundation of China (NSFC)91753205 China
National Natural Science Foundation of China (NSFC)21778051 China
Ministry of Science and Technology (MoST, China)2017YFA0505200 China
National Natural Science Foundation of China (NSFC)21532004 China
Ministry of Science and Technology (MoST, China)2017YFA0505201 China
Ministry of Science and Technology (MoST, China)2016YFA0400903 China
Ministry of Science and Technology (MoST, China)2017YFA0505403 China
CitationJournal: Elife / Year: 2020
Title: Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1.
Authors: Demeng Sun / Sanling Liu / Siyu Li / Mengge Zhang / Fan Yang / Ming Wen / Pan Shi / Tao Wang / Man Pan / Shenghai Chang / Xing Zhang / Longhua Zhang / Changlin Tian / Lei Liu /
Abstract: Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake ...Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism.
History
DepositionJun 28, 2020-
Header (metadata) releaseOct 21, 2020-
Map releaseOct 21, 2020-
UpdateOct 21, 2020-
Current statusOct 21, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0103
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0103
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7cfs
  • Surface level: 0.0103
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30346.png

Downloads & links

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Map

FileDownload / File: emd_30346.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.014 Å
Density
Contour LevelBy AUTHOR: 0.0103 / Movie #1: 0.0103
Minimum - Maximum-0.09515906 - 0.14887664
Average (Standard dev.)0.00026546195 (±0.0038240782)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 202.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z202.800202.800202.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ304304304
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0950.1490.000

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Supplemental data

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Sample components

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Entire : hASIC1a

EntireName: hASIC1a
Components
  • Complex: hASIC1a
    • Protein or peptide: Acid-sensing ion channel 1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: SODIUM IONSodium

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Supramolecule #1: hASIC1a

SupramoleculeName: hASIC1a / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: The optimized coding DNAs for human hASIC1a (Uniprot: P78348) was synthesized by GeneScript. The truncated hASIC1a (with the carboxyl terminal 60 residues removed, named as hASIC1a-DeltaC) ...Details: The optimized coding DNAs for human hASIC1a (Uniprot: P78348) was synthesized by GeneScript. The truncated hASIC1a (with the carboxyl terminal 60 residues removed, named as hASIC1a-DeltaC) was cloned into the pFastBac1 vector (Invitrogen) with 8-His tag at the amino terminus. Baculovirus-infected Sf9 cells (Thermo Fisher) were used for overexpression and were grown at 300K in serum-free SIM SF medium (Sino Biological Inc.).
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Recombinant strain: Sf9

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Macromolecule #1: Acid-sensing ion channel 1

MacromoleculeName: Acid-sensing ion channel 1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.701297 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHHHM ELKAEEEEVG GVQPVSIQAF ASSSTLHGLA HIFSYERLSL KRALWALCFL GSLAVLLCVC TERVQYYFHY HHVTKLDEV AASQLTFPAV TLCNLNEFRF SQVSKNDLYH AGELLALLNN RYEIPDTQMA DEKQLEILQD KANFRSFKPK P FNMREFYD ...String:
MHHHHHHHHM ELKAEEEEVG GVQPVSIQAF ASSSTLHGLA HIFSYERLSL KRALWALCFL GSLAVLLCVC TERVQYYFHY HHVTKLDEV AASQLTFPAV TLCNLNEFRF SQVSKNDLYH AGELLALLNN RYEIPDTQMA DEKQLEILQD KANFRSFKPK P FNMREFYD RAGHDIRDML LSCHFRGEVC SAEDFKVVFT RYGKCYTFNS GRDGRPRLKT MKGGTGNGLE IMLDIQQDEY LP VWGETDE TSFEAGIKVQ IHSQDEPPFI DQLGFGVAPG FQTFVACQEQ RLIYLPPPWG TCKAVTMDSD LDFFDSYSIT ACR IDCETR YLVENCNCRM VHMPGDAPYC TPEQYKECAD PALDFLVEKD QEYCVCEMPC NLTRYGKELS MVKIPSKASA KYLA KKFNK SEQYIGENIL VLDIFFEVLN YETIEQKKAY EIAGLLGDIG GQMGLFIGAS ILTVLELFDY AYEVIKHKLC RR

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 6 / 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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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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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

Concentration2.7 mg/mL
BufferpH: 8
Details: 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: Purified hASIC1a-DeltaC (3 ul) at a concentration of 2.7 mg/ml was added to the freshly plasma-cleaned holey carbon grid (Quantifol, R1.2/1.3, 300 mesh, Cu), blotted for 5 s at 100% humidity ...Details: Purified hASIC1a-DeltaC (3 ul) at a concentration of 2.7 mg/ml was added to the freshly plasma-cleaned holey carbon grid (Quantifol, R1.2/1.3, 300 mesh, Cu), blotted for 5 s at 100% humidity with a Vitrobot Mark IV (ThermoFisher Scientific) and plunge frozen into liquid ethane cooled by liquid nitrogen..
DetailsThe eluted protein in Ni-NTA affinity chromatography was further purified by size-exclusion chromatography in 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS using a Superdex200 10/300GL column (GE HealthCare).The protein was concentrated to about 5 mg/ml based on A280 measurement, using a 100-kDa cutoff Centricon (Millipore).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsGrids were transferred to a Titan Krios electron microscope (FEI) operated at 300 kV equipped with a Gatan K2 Summit direct detection camera. Images of hASIC1a was collected using the automated image acquisition software SerialEM in counting mode with 29,000x magnification yielding a pixel size of 1.014 A. The total dose of 50 e-/A2 was fractionated to 40 frames with 0.2 s per frame. Nominal defocus values ranged from -1.8 to -2.5 um. The dataset of hASIC1a included 3,235 micrographs.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 3235 / Average exposure time: 8.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 274796
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final 3D classificationNumber classes: 213605 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 122890

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Atomic model buiding 1

Initial modelPDB ID:

6ave


Chain - Chain ID: A / Chain - Residue range: 13-462
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7cfs:
Cryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0

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