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Yorodumi- EMDB-30347: Cryo-EM strucutre of human acid-sensing ion channel 1a in complex... -
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-Basic information
Entry | Database: EMDB / ID: EMD-30347 | |||||||||||||||||||||||||||
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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 | |||||||||||||||||||||||||||
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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 / 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 | single particle reconstruction / cryo EM / Resolution: 3.9 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_30347.map.gz | 2.8 MB | EMDB map data format | |
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Header (meta data) | emd-30347-v30.xml emd-30347.xml | 17 KB 17 KB | Display Display | EMDB header |
Images | emd_30347.png | 173.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-30347 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-30347 | HTTPS FTP |
-Validation report
Summary document | emd_30347_validation.pdf.gz | 333.1 KB | Display | EMDB validaton report |
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Full document | emd_30347_full_validation.pdf.gz | 332.7 KB | Display | |
Data in XML | emd_30347_validation.xml.gz | 5.7 KB | Display | |
Data in CIF | emd_30347_validation.cif.gz | 6.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30347 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30347 | HTTPS FTP |
-Related structure data
Related structure data | 7cftMC 7cfsC 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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Related items in Molecule of the Month |
-Map
File | Download / File: emd_30347.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-Mamba1
Entire | Name: hASIC1a-Mamba1 |
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Components |
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-Supramolecule #1: hASIC1a-Mamba1
Supramolecule | Name: hASIC1a-Mamba1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 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. |
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Source (natural) | Organism: Homo sapiens (human) |
-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: Mambalgin-1
Macromolecule | Name: Mambalgin-1 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Dendroaspis polylepis polylepis (black mamba) |
Molecular weight | Theoretical: 6.574644 KDa |
Sequence | String: LKCYQHGKVV TCHRDMKFCY HNTGMPFRNL KLILQGCSSS CSETENNKCC STDRCNK |
-Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 6 / Formula: NAG |
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Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3.1 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-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.. |
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. |
-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-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. |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 3 / Number real images: 3364 / 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 |