+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 7cft | |||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
タイトル | Cryo-EM strucutre of human acid-sensing ion channel 1a in complex with snake toxin Mambalgin1 at pH 8.0 | |||||||||||||||||||||||||||
要素 |
| |||||||||||||||||||||||||||
キーワード | MEMBRANE PROTEIN / ASIC / human / Mambalgin1 toxin / cryo-EM structure | |||||||||||||||||||||||||||
機能・相同性 | 機能・相同性情報 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 類似検索 - 分子機能 | |||||||||||||||||||||||||||
生物種 | Homo sapiens (ヒト) Dendroaspis polylepis polylepis (コブラ) | |||||||||||||||||||||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.9 Å | |||||||||||||||||||||||||||
データ登録者 | Sun, D.M. / Liu, S.L. / Li, S.Y. / Yang, F. / Tian, C.L. | |||||||||||||||||||||||||||
資金援助 | 中国, 8件
| |||||||||||||||||||||||||||
引用 | ジャーナル: Elife / 年: 2020 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 |
|
-構造の表示
ムービー |
ムービービューア |
---|---|
構造ビューア | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 7cft.cif.gz | 255.5 KB | 表示 | PDBx/mmCIF形式 |
---|---|---|---|---|
PDB形式 | pdb7cft.ent.gz | 212.5 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 7cft.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 7cft_validation.pdf.gz | 736.6 KB | 表示 | wwPDB検証レポート |
---|---|---|---|---|
文書・詳細版 | 7cft_full_validation.pdf.gz | 748.3 KB | 表示 | |
XML形式データ | 7cft_validation.xml.gz | 41.1 KB | 表示 | |
CIF形式データ | 7cft_validation.cif.gz | 63 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/cf/7cft ftp://data.pdbj.org/pub/pdb/validation_reports/cf/7cft | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
|
---|---|
1 |
|
-要素
#1: タンパク質 | 分子量: 54701.297 Da / 分子数: 3 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: ASIC1, ACCN2, BNAC2 発現宿主: Spodoptera frugiperda (ツマジロクサヨトウ) 参照: UniProt: P78348 #2: タンパク質 | 分子量: 6574.644 Da / 分子数: 3 / 由来タイプ: 合成 由来: (合成) Dendroaspis polylepis polylepis (コブラ) 参照: UniProt: P0DKR6 #3: 糖 | ChemComp-NAG / 研究の焦点であるリガンドがあるか | N | |
---|
-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
---|---|
EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
-試料調製
構成要素 | 名称: hASIC1a-Mamba1 / タイプ: COMPLEX 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.). The mambalgin1 (Mamba1) toxin was obtained using a hydrazide-based chemical synthesis method. Entity ID: #1-#2 / 由来: MULTIPLE SOURCES |
---|---|
分子量 | 実験値: NO |
由来(天然) | 生物種: Homo sapiens (ヒト) |
由来(組換発現) | 生物種: Spodoptera aff. frugiperda 1 BOLD-2017 (蝶・蛾) 株: Sf9 |
緩衝液 | pH: 8 詳細: 20 mM Tris (pH 8.0), 200 mM NaCl, 0.05% DDM, 0.01% CHS |
試料 | 濃度: 3.1 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: 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 ...詳細: 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. |
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 |
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 298 K 詳細: 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% ...詳細: 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. |
-電子顕微鏡撮影
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
---|---|
顕微鏡 | モデル: FEI TITAN KRIOS 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-Mamba1 complex was collected ...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-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. |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: SPOT SCAN |
電子レンズ | モード: BRIGHT FIELD |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 平均露光時間: 8 sec. / 電子線照射量: 50 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 撮影したグリッド数: 3 / 実像数: 3364 |
画像スキャン | 動画フレーム数/画像: 40 |
-解析
ソフトウェア | 名称: PHENIX / バージョン: 1.14_3260: / 分類: 精密化 | ||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EMソフトウェア |
| ||||||||||||||||||||||||||||
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 234742 | ||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.9 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 119901 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT | ||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 6AVE PDB chain-ID: A / Pdb chain residue range: 13-462 |