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基本情報
登録情報 | データベース: EMDB / ID: EMD-30346 | |||||||||||||||||||||||||||
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タイトル | Cryo-EM strucutre of human acid-sensing ion channel 1a at pH 8.0 | |||||||||||||||||||||||||||
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機能・相同性 | ![]() 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 類似検索 - 分子機能 | |||||||||||||||||||||||||||
生物種 | ![]() | |||||||||||||||||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.56 Å | |||||||||||||||||||||||||||
![]() | Sun DM / Liu SL / Li SY / Yang F / Tian CL | |||||||||||||||||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1. 著者: 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 / ![]() 要旨: 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. | |||||||||||||||||||||||||||
履歴 |
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構造の表示
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-検証レポート
文書・要旨 | ![]() | 330.7 KB | 表示 | ![]() |
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マップ
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ボクセルのサイズ | X=Y=Z: 1.014 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
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試料の構成要素
-全体 : hASIC1a
全体 | 名称: hASIC1a |
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要素 |
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-超分子 #1: hASIC1a
超分子 | 名称: hASIC1a / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1 詳細: 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) ...詳細: 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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由来(天然) | 生物種: ![]() |
組換発現 | 生物種: ![]() 組換株: Sf9 |
-分子 #1: Acid-sensing ion channel 1
分子 | 名称: Acid-sensing ion channel 1 / タイプ: protein_or_peptide / ID: 1 / コピー数: 3 / 光学異性体: LEVO |
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由来(天然) | 生物種: ![]() |
分子量 | 理論値: 54.701297 KDa |
組換発現 | 生物種: ![]() ![]() |
配列 | 文字列: MHHHHHHHHM ELKAEEEEVG GVQPVSIQAF ASSSTLHGLA HIFSYERLSL KRALWALCFL GSLAVLLCVC TERVQYYFHY HHVTKLDEV AASQLTFPAV TLCNLNEFRF SQVSKNDLYH AGELLALLNN RYEIPDTQMA DEKQLEILQD KANFRSFKPK P FNMREFYD ...文字列: 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 |
-分子 #2: 2-acetamido-2-deoxy-beta-D-glucopyranose
分子 | 名称: 2-acetamido-2-deoxy-beta-D-glucopyranose / タイプ: ligand / ID: 2 / コピー数: 6 / 式: NAG |
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分子量 | 理論値: 221.208 Da |
Chemical component information | ![]() ChemComp-NAG: |
-分子 #3: CHOLESTEROL HEMISUCCINATE
分子 | 名称: CHOLESTEROL HEMISUCCINATE / タイプ: ligand / ID: 3 / コピー数: 3 / 式: Y01 |
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分子量 | 理論値: 486.726 Da |
Chemical component information | ![]() ChemComp-Y01: |
-分子 #4: SODIUM ION
分子 | 名称: SODIUM ION / タイプ: ligand / ID: 4 / コピー数: 1 |
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分子量 | 理論値: 22.99 Da |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
濃度 | 2.7 mg/mL |
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緩衝液 | pH: 8 詳細: 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS |
グリッド | モデル: Quantifoil R1.2/1.3 / 材質: COPPER / メッシュ: 300 / 前処理 - タイプ: PLASMA CLEANING / 前処理 - 雰囲気: OTHER / 前処理 - 気圧: 101.325 kPa |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 298 K / 装置: FEI VITROBOT MARK IV 詳細: 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 ...詳細: 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.. |
詳細 | 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). |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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詳細 | 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. |
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 2 / 実像数: 3235 / 平均露光時間: 8.0 sec. / 平均電子線量: 50.0 e/Å2 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: SPOT SCAN / 撮影モード: BRIGHT FIELD |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |