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

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Basic information

Entry
Database: PDB / ID: 7cfs
TitleCryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0
ComponentsAcid-sensing ion channel 1
KeywordsMEMBRANE PROTEIN / ASIC / human / resting state / cryo-EM structure
Function / homology
Function and homology information


gated channel activity / sensory perception of sour taste / monoatomic ion-gated 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 ...gated channel activity / sensory perception of sour taste / monoatomic ion-gated 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
CHOLESTEROL HEMISUCCINATE / Acid-sensing ion channel 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.56 Å
AuthorsSun, D.M. / Liu, S.L. / Li, S.Y. / Yang, F. / Tian, C.L.
Funding support China, 8items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31600601 China
National Natural Science Foundation of China (NSFC)21778051 China
National Natural Science Foundation of China (NSFC)91753205 China
National Natural Science Foundation of China (NSFC)21532004 China
Ministry of Science and Technology (MoST, China)2017YFA0505200 China
Ministry of Science and Technology (MoST, China)2016YFA0400903 China
Ministry of Science and Technology (MoST, China)2017YFA0505201 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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Acid-sensing ion channel 1
B: Acid-sensing ion channel 1
C: Acid-sensing ion channel 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,91413
Polymers164,1043
Non-polymers2,81010
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15040 Å2
ΔGint-89 kcal/mol
Surface area59290 Å2

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Components

#1: Protein Acid-sensing ion channel 1 / / ASIC1 / Amiloride-sensitive cation channel 2 / neuronal / Brain sodium channel 2 / BNaC2


Mass: 54701.297 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ASIC1, ACCN2, BNAC2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P78348
#2: 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.: 6
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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
#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
Has ligand of interestN

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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: hASIC1a / Type: COMPLEX
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.).
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Strain: Sf9
Buffer solutionpH: 8
Details: 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS
SpecimenConc.: 2.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The 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 ...Details: The 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).
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
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.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Grids 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 ...Details: Grids 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.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3235
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
4GctfCTF correction
7UCSF Chimeramodel fitting
11RELION2.1classification
12RELION2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 274796
3D reconstructionResolution: 3.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122890 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 6AVE

6ave


Pdb chain-ID: A / Pdb chain residue range: 13-462

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