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基本情報
登録情報 | データベース: PDB / ID: 1o1f | ||||||
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タイトル | MOLECULAR MODELS OF AVERAGED RIGOR CROSSBRIDGES FROM TOMOGRAMS OF INSECT FLIGHT MUSCLE | ||||||
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![]() | CONTRACTILE PROTEIN / ACTIN-MYOSIN COMPLEX IN SITU IN MUSCLE | ||||||
機能・相同性 | ![]() contractile muscle fiber / Striated Muscle Contraction / myosin filament / myosin II complex / myosin complex / cytoskeletal motor activator activity / microfilament motor activity / myofibril / tropomyosin binding / mesenchyme migration ...contractile muscle fiber / Striated Muscle Contraction / myosin filament / myosin II complex / myosin complex / cytoskeletal motor activator activity / microfilament motor activity / myofibril / tropomyosin binding / mesenchyme migration / troponin I binding / myosin heavy chain binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / striated muscle thin filament / actin filament bundle assembly / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / muscle contraction / stress fiber / skeletal muscle tissue development / titin binding / actin filament polymerization / filopodium / actin filament / 加水分解酵素; 酸無水物に作用; 酸無水物に作用・細胞または細胞小器官の運動に関与 / calcium-dependent protein binding / actin filament binding / lamellipodium / cell body / calmodulin binding / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / ATP binding / identical protein binding / cytoplasm 類似検索 - 分子機能 | ||||||
生物種 | ![]() ![]() ![]() ![]() | ||||||
手法 | 電子顕微鏡法 / 電子線トモグラフィー法 / ネガティブ染色法 / 解像度: 70 Å | ||||||
![]() | Chen, L.F. / Winkler, H. / Reedy, M.K. / Reedy, M.C. / Taylor, K.A. | ||||||
![]() | ![]() タイトル: Molecular modeling of averaged rigor crossbridges from tomograms of insect flight muscle. 著者: Li Fan Chen / Hanspeter Winkler / Michael K Reedy / Mary C Reedy / Kenneth A Taylor / ![]() 要旨: Electron tomography, correspondence analysis, molecular model building, and real-space refinement provide detailed 3-D structures for in situ myosin crossbridges in the nucleotide-free state (rigor), ...Electron tomography, correspondence analysis, molecular model building, and real-space refinement provide detailed 3-D structures for in situ myosin crossbridges in the nucleotide-free state (rigor), thought to represent the end of the power stroke. Unaveraged tomograms from a 25-nm longitudinal section of insect flight muscle preserved native structural variation. Recurring crossbridge motifs that repeat every 38.7 nm along the actin filament were extracted from the tomogram and classified by correspondence analysis into 25 class averages, which improved the signal to noise ratio. Models based on the atomic structures of actin and of myosin subfragment 1 were rebuilt to fit 11 class averages. A real-space refinement procedure was applied to quantitatively fit the reconstructions and to minimize steric clashes between domains introduced during the fitting. These combined procedures show that no single myosin head structure can fit all the in situ crossbridges. The validity of the approach is supported by agreement of these atomic models with fluorescent probe data from vertebrate muscle as well as with data from regulatory light chain crosslinking between heads of smooth muscle heavy meromyosin when bound to actin. #1: ![]() タイトル: Real Space Refinement of Acto-Myosin Structures from Sectioned Muscle. 著者: Chen, L.F. / Blanc, E. / Chapman, M.S. / Taylor, K.A. #2: ![]() タイトル: Multivariate Statistical Analysis of Three-Dimensional Cross-Bridge Motifs in Insect Flight Muscle 著者: Winkler, H. / Taylor, K.A. #3: ![]() タイトル: The Use of Electron Tomography for Structural Analysis of Disordered Protein Arrays. 著者: Taylor, K.A. / Tang, J. / Cheng, Y. / Winkler, H. | ||||||
履歴 |
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Remark 999 | SEQUENCE Since the models used to fit the data are not from the same source, the DBREF and the ...SEQUENCE Since the models used to fit the data are not from the same source, the DBREF and the SEQADV remarks were suppressed. |
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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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: 96625.484 Da / 分子数: 4 / 由来タイプ: 天然 詳細: ORGANISM FROM WHICH THE MYOSIN FOR THE CRYSTAL STRUCTURE THAT PROVIDED 2MYS WAS OBTAINED 由来: (天然) ![]() ![]() #2: タンパク質 | 分子量: 16063.036 Da / 分子数: 4 / 由来タイプ: 天然 詳細: ORGANISM FROM WHICH THE MYOSIN FOR THE CRYSTAL STRUCTURE THAT PROVIDED 2MYS WAS OBTAINED 由来: (天然) ![]() ![]() #3: タンパク質 | 分子量: 16063.926 Da / 分子数: 4 / 由来タイプ: 天然 詳細: ORGANISM FROM WHICH THE MYOSIN FOR THE CRYSTAL STRUCTURE THAT PROVIDED 2MYS WAS OBTAINED 由来: (天然) ![]() ![]() #4: タンパク質 | 分子量: 41862.613 Da / 分子数: 14 / 由来タイプ: 天然 詳細: ORGANISM FROM WHICH THE ACTIN THAT PROVIDED THE CRYSTAL STRUCTURE FOR 1ATN WAS OBTAINED 由来: (天然) ![]() ![]() |
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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: TISSUE / 3次元再構成法: 電子線トモグラフィー法 |
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試料調製
構成要素 | 名称: INSECT FLIGHT MUSCLE OF THE LARGE WATER BUG LETHOCERUS MAXIMUS タイプ: TISSUE 詳細: DORSAL LONGITUDINAL FLIGHT MUSCLES OF THE LARGE WATERBUG, LETHOCERUS MAXIMUS WERE GLYCERINATED IN THE DISSECTED THORAX AND STORED AT -20 DEGREES C. FIXATION INVOLVED SEQUENTIAL TANNIC ACID- ...詳細: DORSAL LONGITUDINAL FLIGHT MUSCLES OF THE LARGE WATERBUG, LETHOCERUS MAXIMUS WERE GLYCERINATED IN THE DISSECTED THORAX AND STORED AT -20 DEGREES C. FIXATION INVOLVED SEQUENTIAL TANNIC ACID-GLUTARALDEHYDE (0.2% AND 2%), COLD 1% OSO4 AT PH 6.0, 1% URANYL ACETATE BLOCK STAINING AND ARALDITE 506 EMGEDDING. THIN SECTIONS WERE STAINED WITH A SEQUENCE OF PERMANGANATE AND LEAD. SECTION WERE ~25 NM THICK. MODELS ARE BUILT TO FIT THE THIN, ACTIN CONTAINING FILAMENTS LABELED WITH ENDOGENOUS MYOSIN IN THE RIGOR STATE. |
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試料 | 包埋: YES / シャドウイング: NO / 染色: YES / 凍結: NO |
染色 | タイプ: NEGATIVE / 染色剤: Osmium tetroxide, Uranyl Acetate |
急速凍結 | 詳細: NO VITRIFICATION. SAMPLES WERE VIEWED AT ROOM TEMPERATURE. |
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電子顕微鏡撮影
顕微鏡 | モデル: FEI/PHILIPS EM400 |
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電子銃 | 電子線源: TUNGSTEN HAIRPIN / 加速電圧: 100 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 17000 X |
撮影 | フィルム・検出器のモデル: KODAK SO-163 FILM |
画像スキャン | デジタル画像の数: 36 |
放射 | プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M |
放射波長 | 相対比: 1 |
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解析
EMソフトウェア |
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対称性 | 点対称性: C1 (非対称) | |||||||||||||||||||||
3次元再構成 | 手法: DUAL AXIS TILT SERIES ELECTRON TOMOGRAPHY / 解像度: 70 Å / ピクセルサイズ(公称値): 15.5 Å / 倍率補正: INDICATED INSTRUMENT MAGNIFICATION 詳細: 3-D MOTIFS WERE IDENTIFIED IN THE TOMOGRAM BY FIRST PRODUCING A CROSS CORRELATION MAP FROM WHICH PEAK COORDINATES WERE DETERMINED FROM THEIR CENTER OF GRAVITY. WE DEFINE A 3-D MOTIF AS ONE, ...詳細: 3-D MOTIFS WERE IDENTIFIED IN THE TOMOGRAM BY FIRST PRODUCING A CROSS CORRELATION MAP FROM WHICH PEAK COORDINATES WERE DETERMINED FROM THEIR CENTER OF GRAVITY. WE DEFINE A 3-D MOTIF AS ONE, ENTIRE 38.7 NM CROSSBRIDGE REPEAT ALONG ACTIN. THESE MOTIFS USUALLY CONTAIN AT LEAST FOUR MYOSIN HEADS IN TWO PAIRED CROSSBRIDGES (SINGLE CHEVRONS) AND SOMETIMES CONTAIN AS MANY AS SIX MYOSIN HEADS IN FOUR PAIRED CROSSBRIDGES (DOUBLE CHEVRONS). THE REFERENCE FOR THE ANALYSIS WAS SELECTED TO BE CENTERED BETWEEN SUCCESSIVE TROPONIN DENSITIES WHICH COULD BE IDENTIFIED FROM THE IN-PLANE PROJECTION. THE INDIVIDUAL CROSSBRIDGE MOTIFS WERE THEN SUBJECTED TO MULTIVARIATE STATISTICAL ANALYSIS TO IDENTIFY CLUSTERS OF MOTIFS SHOWING SIMILAR CROSSBRIDGE STRUCTURE. THESE CLUSTERS FORMED THE CLASS AVERAGES. THE CHOICE OF STRUCTURE TO BE CLASSIFIED WAS DECIDED BY THE RESOLUTION AND THE LATER PROCESS OF MODEL BUILDING. AVERAGING WAS DONE ACCORDING TO THE HEIRARCHICAL ASCENDENT METHOD. THE RESOLUTION IN EACH OF THE CLASS AVERAGES WAS 7 NM BY THE SPECTRAL SIGNAL TO NOISE RATIO. 対称性のタイプ: POINT | |||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT / 空間: REAL Target criteria: BEST CORRELATION COEFFICIENT AND FEWEST POOR CONTACTS 詳細: METHOD--INITIAL MODELS WERE FIT BY HAND USING O. THE FIT WAS THEN REFINED USING REAL SPACE REFINEMENT. REFINEMENT PROTOCOL--RIGID BODY | |||||||||||||||||||||
原子モデル構築 |
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精密化 | 最高解像度: 70 Å | |||||||||||||||||||||
精密化ステップ | サイクル: LAST / 最高解像度: 70 Å
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