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Yorodumi- EMDB-30346: Cryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0 -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-30346 | |||||||||||||||||||||||||||
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Title | Cryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0 | |||||||||||||||||||||||||||
Map data | ||||||||||||||||||||||||||||
Sample |
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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 / response to acidic pH / sodium ion transport / protein homotrimerization ...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 / 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 / glutamatergic synapse / Golgi apparatus / cell surface / plasma membrane Similarity search - Function | |||||||||||||||||||||||||||
Biological species | Homo sapiens (human) | |||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.56 Å | |||||||||||||||||||||||||||
Authors | Sun DM / Liu SL / Li SY / Yang F / Tian CL | |||||||||||||||||||||||||||
Funding support | China, 8 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_30346.map.gz | 2.4 MB | EMDB map data format | |
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Header (meta data) | emd-30346-v30.xml emd-30346.xml | 16.9 KB 16.9 KB | Display Display | EMDB header |
Images | emd_30346.png | 157.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-30346 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-30346 | HTTPS FTP |
-Validation report
Summary document | emd_30346_validation.pdf.gz | 330.7 KB | Display | EMDB validaton report |
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Full document | emd_30346_full_validation.pdf.gz | 330.3 KB | Display | |
Data in XML | emd_30346_validation.xml.gz | 5.6 KB | Display | |
Data in CIF | emd_30346_validation.cif.gz | 6.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30346 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30346 | HTTPS FTP |
-Related structure data
Related structure data | 7cfsMC 7cftC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_30346.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.014 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : hASIC1a
Entire | Name: hASIC1a |
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Components |
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-Supramolecule #1: hASIC1a
Supramolecule | Name: 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.). |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths) Recombinant strain: Sf9 |
-Macromolecule #1: Acid-sensing ion channel 1
Macromolecule | Name: Acid-sensing ion channel 1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 54.701297 KDa |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | String: 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 |
-Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 6 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Macromolecule #3: CHOLESTEROL HEMISUCCINATE
Macromolecule | Name: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 3 / Number of copies: 3 / Formula: Y01 |
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Molecular weight | Theoretical: 486.726 Da |
Chemical component information | ChemComp-Y01: |
-Macromolecule #4: SODIUM ION
Macromolecule | Name: SODIUM ION / type: ligand / ID: 4 / Number of copies: 1 |
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Molecular weight | Theoretical: 22.99 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2.7 mg/mL |
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Buffer | pH: 8 Details: 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 101.325 kPa |
Vitrification | Cryogen 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.. |
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). |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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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. |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |