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Yorodumi- PDB-7cft: Cryo-EM strucutre of human acid-sensing ion channel 1a in complex... -
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Entry | Database: PDB / ID: 7cft | |||||||||||||||||||||||||||
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Title | Cryo-EM strucutre of human acid-sensing ion channel 1a in complex with snake toxin Mambalgin1 at pH 8.0 | |||||||||||||||||||||||||||
Components |
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Keywords | MEMBRANE PROTEIN / ASIC / human / Mambalgin1 toxin / cryo-EM structure | |||||||||||||||||||||||||||
Function / homology | Function and homology information sensory perception of sour taste / monoatomic ion-gated channel activity / pH-gated monoatomic ion channel activity / ligand-gated sodium channel activity / cellular response to pH / negative regulation of neurotransmitter secretion / neurotransmitter secretion / ion channel regulator activity / response to acidic pH / sodium ion transport ...sensory perception of sour taste / monoatomic ion-gated channel activity / pH-gated monoatomic ion channel activity / ligand-gated sodium channel activity / cellular response to pH / negative regulation of neurotransmitter secretion / neurotransmitter secretion / ion channel regulator activity / response to acidic pH / sodium ion transport / protein homotrimerization / associative learning / behavioral fear response / sodium ion transmembrane transport / response to amphetamine / regulation of membrane potential / calcium ion transmembrane transport / postsynaptic density membrane / Stimuli-sensing channels / memory / presynapse / toxin activity / glutamatergic synapse / Golgi apparatus / cell surface / extracellular region / plasma membrane Similarity search - Function | |||||||||||||||||||||||||||
Biological species | Homo sapiens (human) Dendroaspis polylepis polylepis (black mamba) | |||||||||||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||||||||||||||||||||
Authors | Sun, D.M. / Liu, S.L. / Li, S.Y. / Yang, F. / Tian, C.L. | |||||||||||||||||||||||||||
Funding support | China, 8items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7cft.cif.gz | 255.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7cft.ent.gz | 212.5 KB | Display | PDB format |
PDBx/mmJSON format | 7cft.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7cft_validation.pdf.gz | 736.6 KB | Display | wwPDB validaton report |
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Full document | 7cft_full_validation.pdf.gz | 748.3 KB | Display | |
Data in XML | 7cft_validation.xml.gz | 41.1 KB | Display | |
Data in CIF | 7cft_validation.cif.gz | 63 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/cf/7cft ftp://data.pdbj.org/pub/pdb/validation_reports/cf/7cft | HTTPS FTP |
-Related structure data
Related structure data | 30347MC 7cfsC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | 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: Protein | Mass: 6574.644 Da / Num. of mol.: 3 / Source method: obtained synthetically Source: (synth.) Dendroaspis polylepis polylepis (black mamba) References: UniProt: P0DKR6 #3: Sugar | ChemComp-NAG / Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: hASIC1a-Mamba1 / 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.). The mambalgin1 (Mamba1) toxin was obtained using a hydrazide-based chemical synthesis method. Entity ID: #1-#2 / Source: MULTIPLE SOURCES |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) Strain: Sf9 |
Buffer solution | pH: 8 Details: 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS |
Specimen | Conc.: 3.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The purified hASIC1a protein in a buffer comtaining 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS concentrated to about 5 mg/ml based on A280 measurement, using a 100-kDa cutoff ...Details: The purified hASIC1a protein in a buffer comtaining 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS concentrated to about 5 mg/ml based on A280 measurement, using a 100-kDa cutoff Centricon (Millipore). The chemical synthesized, lyophilized Mamba1 was dissolved in buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS at a final concentration of 10 mg/ml based on A280 measurement, and added in a 6:1 molar ratio of toxin to hASIC1a channel with incubation for 1 h at 297K. |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K Details: Purified hASIC1a-Mamba1 complex (3 ul) at a concentration of 3.1 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% ...Details: Purified hASIC1a-Mamba1 complex (3 ul) at a concentration of 3.1 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: 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-Mamba1 complex was collected ...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-Mamba1 complex 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 3,364 micrographs. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average 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: 3 / Num. of real images: 3364 |
Image scans | Movie frames/image: 40 |
-Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement | ||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 234742 | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119901 / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6AVE Pdb chain-ID: A / Pdb chain residue range: 13-462 |