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基本情報
登録情報 | データベース: EMDB / ID: EMD-1950 | ||||||||||||
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タイトル | 3D-Structure of tarantula myosin filament obtained by cryo-electron microscopy | ||||||||||||
![]() | This is a density map of tarantula thick filaments, the initial view is from the Z line perspective, if the map is rotated by 90 degress in x direction, the J motif of the interacting heads features and the backbone subfilaments can be seen clearly | ||||||||||||
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![]() | cryo-EM / thick filament / flexible docking / single particle reconstruction / Iterative Helical Real Space Reconstruction (IHRSR) / Myosin regulation / myosin regulatory light chain / phosphorylation | ||||||||||||
機能・相同性 | ![]() myosin II filament / regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / myofibril assembly / elastic fiber assembly / myosin light chain binding / skeletal muscle myosin thick filament assembly / myosin II binding / muscle myosin complex / muscle filament sliding ...myosin II filament / regulation of slow-twitch skeletal muscle fiber contraction / regulation of the force of skeletal muscle contraction / myofibril assembly / elastic fiber assembly / myosin light chain binding / skeletal muscle myosin thick filament assembly / myosin II binding / muscle myosin complex / muscle filament sliding / transition between fast and slow fiber / regulation of the force of heart contraction / actomyosin / myosin filament / adult heart development / cardiac muscle hypertrophy in response to stress / actomyosin structure organization / myosin II complex / myosin complex / cardiac muscle cell development / structural constituent of muscle / ventricular cardiac muscle tissue morphogenesis / sarcomere organization / microfilament motor activity / myofibril / myosin heavy chain binding / cytoskeletal motor activity / skeletal muscle contraction / smooth muscle contraction / striated muscle contraction / muscle contraction / cardiac muscle contraction / stress fiber / ATP metabolic process / regulation of heart rate / sarcomere / ADP binding / Z disc / actin filament binding / actin binding / calmodulin binding / calcium ion binding / magnesium ion binding / ATP binding / cytosol / cytoplasm 類似検索 - 分子機能 | ||||||||||||
生物種 | ![]() | ||||||||||||
手法 | らせん対称体再構成法 / クライオ電子顕微鏡法 / ネガティブ染色法 / 解像度: 20.0 Å | ||||||||||||
![]() | Alamo L / Wriggers W / Pinto A / Bartoli F / Salazar L / Zhao F / Craig R / Padron R | ||||||||||||
![]() | ![]() タイトル: Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity. 著者: Lorenzo Alamo / Willy Wriggers / Antonio Pinto / Fulvia Bártoli / Leiria Salazar / Fa-Qing Zhao / Roger Craig / Raúl Padrón / ![]() 要旨: Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular ...Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament. | ||||||||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | EMマップ: ![]() ![]() ![]() |
添付画像 |
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-EMDBアーカイブ
マップデータ | ![]() | 46.9 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 15.7 KB 15.7 KB | 表示 表示 | ![]() |
画像 | ![]() | 107.9 KB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
関連構造データ | ![]() 3dtpMU ![]() 3jbhM M: このマップから作成された原子モデル U: あてはまらない、座標系が異なる*YM |
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類似構造データ | 類似検索 - 機能・相同性 ![]() |
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リンク
EMDBのページ | ![]() ![]() |
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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | This is a density map of tarantula thick filaments, the initial view is from the Z line perspective, if the map is rotated by 90 degress in x direction, the J motif of the interacting heads features and the backbone subfilaments can be seen clearly | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X: 2.48 Å / Y: 2.48 Å / Z: 2.482 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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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 : Myosin filaments from Tarantula striated muscle
全体 | 名称: Myosin filaments from Tarantula striated muscle |
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要素 |
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-超分子 #1000: Myosin filaments from Tarantula striated muscle
超分子 | 名称: Myosin filaments from Tarantula striated muscle / タイプ: sample / ID: 1000 集合状態: Polymer of a multiple myosin assembled over a paramyosin core Number unique components: 2 |
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-分子 #1: Myosin II
分子 | 名称: Myosin II / タイプ: protein_or_peptide / ID: 1 / Name.synonym: Myosin Type II / 集合状態: Polymer / 組換発現: No / データベース: NCBI |
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由来(天然) | 生物種: ![]() |
-実験情報
-構造解析
手法 | ネガティブ染色法, クライオ電子顕微鏡法 |
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![]() | らせん対称体再構成法 |
試料の集合状態 | filament |
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試料調製
緩衝液 | pH: 7 詳細: 100mM NaCl,3mM MgCl2,1mM EGTA, 5mM PIPES, 5mM NaH2PO4,1mM NaN3. |
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染色 | タイプ: NEGATIVE 詳細: A 6 ul aliquot of native purified tarantula thick filaments suspension (Hidalgo et al. 2001) was applied to a 400 mesh grid coated with a holey carbon film that had been rendered hydrophilic ...詳細: A 6 ul aliquot of native purified tarantula thick filaments suspension (Hidalgo et al. 2001) was applied to a 400 mesh grid coated with a holey carbon film that had been rendered hydrophilic by glow discharge in n-amylamine vapor for 3 minutes before use. After allowing the filaments to adsorb to the grid for 30 seconds, the grid was rinsed with the relaxing rinse, then placed in a humidity chamber (aprox. 80% relative humidity). Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen. |
グリッド | 詳細: Holey carbon grids 400 mesh |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 80 % / チャンバー内温度: 93 K / 装置: HOMEMADE PLUNGER 詳細: Vitrification instrument: Home-made plunger. Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately ...詳細: Vitrification instrument: Home-made plunger. Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen. 手法: Plunging in a liquid ethane |
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電子顕微鏡法
顕微鏡 | FEI/PHILIPS CM120T |
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温度 | 最低: 88 K / 最高: 90 K |
詳細 | Holey carbon grids Cryo preserved in Liquid ethane were observed in a Philips CM120 electron microscope under low dose conditions. Only filaments on thin carbon over holes were photographed |
日付 | 2002年10月23日 |
撮影 | カテゴリ: FILM / フィルム・検出器のモデル: KODAK SO-163 FILM / デジタル化 - スキャナー: OTHER / デジタル化 - サンプリング間隔: 8.47 µm / 実像数: 1008 / ビット/ピクセル: 14 |
電子線 | 加速電圧: 120 kV / 電子線源: LAB6 |
電子光学系 | 倍率(補正後): 35000 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.0 mm / 最大 デフォーカス(公称値): 1.95 µm / 最小 デフォーカス(公称値): 1.95 µm / 倍率(公称値): 35000 |
試料ステージ | 試料ホルダー: Eucentric / 試料ホルダーモデル: GATAN LIQUID NITROGEN |
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画像解析
詳細 | There are 4 helices of myosin heads, rotated 30 degrees, every 145 Angstroms. The filament segments were selected based on visual judgement of good helical order |
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最終 再構成 | 想定した対称性 - らせんパラメータ - Δz: 100 Å 想定した対称性 - らせんパラメータ - ΔΦ: 30 ° 想定した対称性 - らせんパラメータ - 軸対称性: C4 (4回回転対称) アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 20.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: SPIDER 詳細: Three-dimensional single particle reconstruction was carried out by a modification of the IHRSR method, using SPIDER. Low-dose electron micrographs of 1008 frozen-hydrated thick filaments ...詳細: Three-dimensional single particle reconstruction was carried out by a modification of the IHRSR method, using SPIDER. Low-dose electron micrographs of 1008 frozen-hydrated thick filaments halves ere digitized at 0.248 nm per pixel using a Nikon Super Coolscan 8000 ED scanner. Filaments were aligned with the bare zone at the top, to ensure correct polarity in subsequent steps. A total of 15,504 segments, each 62 nm long, with an overlap of 55.8 nm, and containing aprox. 40,000 unique pairs of interacting myosin heads went into the reconstruction. As an initial reference model we used the tarantula negatively stained 3D-map, which was axially rotated, axially shifted and also out of plane tilted up to plus-minus12deg. for projection matching, giving a total of 4,095 projections (13 tilted projections plus-minus12deg. every 2deg., 45 reference rotated projections (0-90 degrees, 2deg. rotation angle), and 7 image axial shifts of 2.2 nm. The resulting 3D-map combines about 10,700 out of 15,504 filament segments, a yield of 69 percent of included segments. |
-原子モデル構築 1
初期モデル | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F |
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ソフトウェア | 名称: Situs 2.3 |
詳細 | PDBEntryID_givenInChain. Protocol: Flexible Fitting. The flexible docking procedure is based on a connected (motion capture) network of identified features within the atomic model. The atomic model is allowed to move according to displacements tracked by 31 control points defined by the network, in order to find the best match to the cryo-EM map |
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT / 当てはまり具合の基準: Correlation |
得られたモデル | ![]() PDB-3dtp: ![]() PDB-3jbh: |