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基本情報
登録情報 | データベース: PDB / ID: 5nl2 | |||||||||
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タイトル | cryo-EM structure of the mTMEM16A ion channel at 6.6 A resolution. | |||||||||
![]() | Anoctamin-1 | |||||||||
![]() | MEMBRANE PROTEIN / TMEM16 family / ion channel / cryo-EM | |||||||||
機能・相同性 | ![]() glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / voltage-gated chloride channel activity / Stimuli-sensing channels / chloride transport ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / voltage-gated chloride channel activity / Stimuli-sensing channels / chloride transport / chloride channel activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / detection of temperature stimulus involved in sensory perception of pain / chloride channel complex / chloride transmembrane transport / regulation of membrane potential / establishment of localization in cell / cell projection / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / cellular response to heat / apical plasma membrane / external side of plasma membrane / signaling receptor binding / glutamatergic synapse / protein homodimerization activity / identical protein binding / metal ion binding / plasma membrane 類似検索 - 分子機能 | |||||||||
生物種 | ![]() ![]() | |||||||||
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: 6.6 Å | |||||||||
![]() | Paulino, C. / Neldner, Y. / Lam, K.M. / Kalienkova, V. / Brunner, J.D. / Schenck, S. / Dutzler, R. | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A. 著者: Cristina Paulino / Yvonne Neldner / Andy Km Lam / Valeria Kalienkova / Janine Denise Brunner / Stephan Schenck / Raimund Dutzler / ![]() 要旨: The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has ...The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family. | |||||||||
履歴 |
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 151.3 KB | 表示 | ![]() |
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PDB形式 | ![]() | 91.6 KB | 表示 | ![]() |
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-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 111058.992 Da / 分子数: 2 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() 細胞株 (発現宿主): HEK293 stable mTMEM16A cell line (Flp-In System) 発現宿主: ![]() |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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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 |
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試料調製
構成要素 | 名称: mTMEM16A / タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT |
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分子量 | 値: 0.110916 MDa / 実験値: YES |
由来(天然) | 生物種: ![]() ![]() |
由来(組換発現) | 生物種: ![]() |
緩衝液 | pH: 7.5 / 詳細: 20 mM Hepes 150 mM NaCl 0.5 mM CaCl2 <0.12% digitonin |
緩衝液成分 | 濃度: 20 mM / 名称: Hepes |
試料 | 濃度: 3.08 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: full-length (wild-type isoform ac) deglycosylated mTMEM16A |
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 200 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: 293 K / 詳細: 2.5 ul sample volume 1-5 sec blotting time |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 37037 X / 倍率(補正後): 37037 X / 最大 デフォーカス(公称値): 3800 nm / 最小 デフォーカス(公称値): 500 nm / Calibrated defocus min: 500 nm / 最大 デフォーカス(補正後): 3800 nm / Cs: 2.7 mm / C2レンズ絞り径: 100 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 最高温度: 100 K / 最低温度: 80 K |
撮影 | 平均露光時間: 15 sec. / 電子線照射量: 80 e/Å2 / 検出モード: SUPER-RESOLUTION 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: 5 / 実像数: 5503 詳細: Data were collected in an automated fashion on a K2 Summit detector (Gatan) set to super-resolution mode with a pixel size of 0.675 A and a defocus range of -0.5 to -3.8 um using SerialEM. ...詳細: Data were collected in an automated fashion on a K2 Summit detector (Gatan) set to super-resolution mode with a pixel size of 0.675 A and a defocus range of -0.5 to -3.8 um using SerialEM. Images were recorded for 15 sec with an initial sub-frame exposure time of 300 ms (50 frames total) with a dose of 1.5 e-/A^2/frame, and later with a sub-frame exposure time of 150 ms (100 frames total) with a dose of 0.8 e-/A^2/frame, resulting in a total accumulated dose on the specimen level of approximately 80 e-/A^2. |
電子光学装置 | エネルギーフィルター名称: GIF / エネルギーフィルター 上限: 10 eV / エネルギーフィルター 下限: -10 eV |
画像スキャン | サンプリングサイズ: 5 µm / 横: 7420 / 縦: 7676 / 動画フレーム数/画像: 50 / 利用したフレーム数/画像: 1-50 |
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解析
ソフトウェア | 名称: PHENIX / バージョン: 1.11.1_2575: / 分類: 精密化 | ||||||||||||||||||||||||||||||||||||||||
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EMソフトウェア |
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画像処理 | 詳細: Fourier cropping (final pixel size 1.35 A), motion correction and dose-weighting of frames were performed by MotionCor2 | ||||||||||||||||||||||||||||||||||||||||
CTF補正 | 詳細: The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1 タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 755348 詳細: Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, resulting in 4,178 images used for further analysis. Image processing was performed using ...詳細: Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, resulting in 4,178 images used for further analysis. Image processing was performed using the software package RELION1.4 and at a later stage RELION2.0. Approximately 4,000 particles were manually picked to generate templates for automated particle selection. Following automated picking in RELION, false positives were eliminated manually or through a first round of 2D classification resulting in 755,348 particles. | ||||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C2 (2回回転対称) | ||||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 6.6 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 213243 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT | ||||||||||||||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 4WIT Accession code: 4WIT / Source name: PDB / タイプ: experimental model | ||||||||||||||||||||||||||||||||||||||||
拘束条件 |
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