+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 6eo1 | ||||||
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タイトル | The electron crystallography structure of the cAMP-bound potassium channel MloK1 (PCO-refined) | ||||||
要素 | Cyclic nucleotide-gated potassium channel mll3241 | ||||||
キーワード | MEMBRANE PROTEIN / MloK1 / MlotiK1 / potassium channel / CNBD / cytoplasmic domains / PCO refinement | ||||||
機能・相同性 | 機能・相同性情報 potassium channel activity / cAMP binding / protein-containing complex binding / identical protein binding / plasma membrane 類似検索 - 分子機能 | ||||||
生物種 | Mesorhizobium loti MAFF303099 (根粒菌) | ||||||
手法 | 電子線結晶学 / クライオ電子顕微鏡法 / 解像度: 4.5 Å | ||||||
データ登録者 | Kowal, J. / Biyani, N. / Chami, M. / Scherer, S. / Rzepiela, A. / Baumgartner, P. / Upadhyay, V. / Nimigean, C. / Stahlberg, H. | ||||||
資金援助 | スイス, 1件
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引用 | ジャーナル: Structure / 年: 2018 タイトル: High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer. 著者: Julia Kowal / Nikhil Biyani / Mohamed Chami / Sebastian Scherer / Andrzej J Rzepiela / Paul Baumgartner / Vikrant Upadhyay / Crina M Nimigean / Henning Stahlberg / 要旨: Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a ...Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a structural model for the cyclic nucleotide-modulated potassium channel homolog from Mesorhizobium loti, MloK1, determined from 2D crystals in the presence of lipids. Even though crystals diffract electrons to only ∼10 Å, using cryoelectron microscopy (cryo-EM) and recently developed computational methods, we have determined a 3D map of full-length MloK1 in the presence of cyclic AMP (cAMP) at ∼4.5 Å isotropic 3D resolution. The structure provides a clear picture of the arrangement of the cyclic nucleotide-binding domains with respect to both the pore and the putative voltage sensor domains when cAMP is bound, and reveals a potential gating mechanism in the context of the lipid-embedded channel. | ||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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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 | 6eo1.cif.gz | 325.8 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb6eo1.ent.gz | 272 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 6eo1.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 | 6eo1_validation.pdf.gz | 896.4 KB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 6eo1_full_validation.pdf.gz | 1 MB | 表示 | |
XML形式データ | 6eo1_validation.xml.gz | 68.5 KB | 表示 | |
CIF形式データ | 6eo1_validation.cif.gz | 95.6 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/eo/6eo1 ftp://data.pdbj.org/pub/pdb/validation_reports/eo/6eo1 | HTTPS FTP |
-関連構造データ
-リンク
-集合体
登録構造単位 |
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単位格子 |
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-要素
#1: タンパク質 | 分子量: 37766.297 Da / 分子数: 4 / 由来タイプ: 組換発現 由来: (組換発現) Mesorhizobium loti MAFF303099 (根粒菌) 遺伝子: mll3241 / 器官: Membrane / プラスミド: pASK90 / Cell (発現宿主): E. coli / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 参照: UniProt: Q98GN8 #2: 化合物 | |
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-実験情報
-実験
実験 | 手法: 電子線結晶学 |
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EM実験 | 試料の集合状態: 2D ARRAY / 3次元再構成法: 電子線結晶学 |
結晶の対称性 | ∠γ: 90 ° / C sampling length: 135 Å / A: 135 Å / B: 135 Å / C: 200 Å / Space group name H-M: P4212 |
-試料調製
構成要素 | 名称: MloK1 tetramer / タイプ: COMPLEX 詳細: Cyclic nucleotide-modulated potassium channel in the presence of cAMP ligand, reconstituted into 2D lipid membrane crystals. Entity ID: #1 / 由来: RECOMBINANT |
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分子量 | 値: 0.148 MDa / 実験値: NO |
由来(天然) | 生物種: Mesorhizobium loti (根粒菌) / 器官: Membrane |
由来(組換発現) | 生物種: Escherichia coli BL21(DE3) (大腸菌) / プラスミド: pASK90 |
EM crystal formation | 装置: dialysis buttons / Atmosphere: dialysis buffer 詳細: DM solubilized MloK1 sample was mixed with E. coli polar lipid extract (Avanti Polar Lipids) at a lipid to protein ratio of 0.8 and dialyzed against detergent free buffer. 2D crystals of the ...詳細: DM solubilized MloK1 sample was mixed with E. coli polar lipid extract (Avanti Polar Lipids) at a lipid to protein ratio of 0.8 and dialyzed against detergent free buffer. 2D crystals of the lipid embedded protein were obtained within 5 days. Lipid mixture: E.coli polar lipids / Lipid protein ratio: 0.8 / 温度: 293 K / Time: 5 DAY |
緩衝液 | pH: 7.6 詳細: 20 mM KCl, 20 mM Tris-HCl pH 7.6, 1 mM BaCl2, 1 mM EDTA, 0.2 mM cAMP |
試料 | 濃度: 0.7 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R3.5/1 |
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 90 % / 凍結前の試料温度: 293 K / 詳細: 3.5 second-blotting |
-データ収集
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 / 詳細: pixel size 1.3 A/pix |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 50000 X / 最大 デフォーカス(公称値): 4300 nm / 最小 デフォーカス(公称値): 750 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 |
撮影 | 平均露光時間: 16 sec. / 電子線照射量: 45 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: 30 / 実像数: 346 詳細: Each image was dose-fractionated in 40 frames (16 sec in total, 0.4-sec frames). The dose rate was set to ~5 counts/sec/physical-pixel (~2.8 e-/s/A2)leading to a total dose of ~45 e-/A2. Pixel size was 1.3A/pix. |
電子光学装置 | エネルギーフィルター名称: GIF Quantum LS / エネルギーフィルター 上限: 20 eV / エネルギーフィルター 下限: 0 eV |
画像スキャン | 動画フレーム数/画像: 40 |
EM回折 | カメラ長: 800 mm / Tilt angle list: 0, 55 |
EM回折 シェル | 解像度: 4.5→6 Å / フーリエ空間範囲: 100 % / 多重度: 300 / 構造因子数: 6901357 / 位相残差: 99 ° |
EM回折 統計 | 詳細: Image processing was done with FOCUS (formerly 2dx), available at FOCUS-EM.org. Arheit et al., 2013a; Arheit et al., 2013b; Arheit et al., 2013c; Gipson et al., 2008; Gipson et al., 2007; ...詳細: Image processing was done with FOCUS (formerly 2dx), available at FOCUS-EM.org. Arheit et al., 2013a; Arheit et al., 2013b; Arheit et al., 2013c; Gipson et al., 2008; Gipson et al., 2007; Gipson et al., 2011; Biyani et al., 2017. Rsym, Rmerge, and phase errors are not available. フーリエ空間範囲: 100 % / 再高解像度: 4.5 Å / 測定した強度の数: 6361 / 構造因子数: 6901357 / 位相誤差: 99 ° / 位相残差: 99 ° / 位相誤差の除外基準: 99 / Rmerge: 99 / Rsym: 99 |
-解析
ソフトウェア | 名称: PHENIX / バージョン: 1.10.1_2155: / 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: Gatan Quantum-LS energy filter, with K2 Summit detector | |||||||||||||||||||||||||
結晶の対称性 | ∠γ: 90 ° / C sampling length: 135 Å / A: 135 Å / B: 135 Å / C: 200 Å / Space group name H-M: P4212 | |||||||||||||||||||||||||
CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||
3次元再構成 | 手法: CRYSTALLOGRAPHY / 解像度: 4.5 Å / 解像度の算出法: OTHER / 対称性のタイプ: 2D CRYSTAL | |||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: fit energy 詳細: The initial model was obtained using Modeller (Sali and Blundell, 1993); in particular, the missing fragments were generated for the previously published PDB 4CHV model. This starting model ...詳細: The initial model was obtained using Modeller (Sali and Blundell, 1993); in particular, the missing fragments were generated for the previously published PDB 4CHV model. This starting model was refined using the Rosetta for cryo-EM package (DiMaio et al., 2015). The symmetry of the channel was restrained during optimization runs (performed following the package tutorial (Wang and DiMaio,2015)). A model with a high fit score to the cryo-EM map and a low energy, as defined by the Rosetta force field, was selected from 100 Rosetta models generated and refined further. Several rounds of manual refinement with Coot (Emsley et al.,2010) and global optimization with Phenix (real_space_refine method (Afonine et al., 2013)) were carried out. Secondary structure constraints were imposed to stabilize the fold of helices and b-sheets during the global optimization. | |||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 4CHV PDB chain-ID: A / Accession code: 4CHV / Pdb chain residue range: 1-355 / Source name: PDB / タイプ: experimental model | |||||||||||||||||||||||||
精密化 | 解像度: 4.5→6 Å / 交差検証法: NONE /
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拘束条件 |
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