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基本情報
登録情報 | データベース: PDB / ID: 2w49 | ||||||
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タイトル | ISOMETRICALLY CONTRACTING INSECT ASYNCHRONOUS FLIGHT MUSCLE | ||||||
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![]() | CONTRACTILE PROTEIN / ISOMETRIC CONTRACTION | ||||||
機能・相同性 | ![]() troponin C binding / troponin complex / Striated Muscle Contraction / regulation of muscle contraction / cytoskeletal motor activator activity / myosin II complex / sarcomere organization / tropomyosin binding / mesenchyme migration / troponin I binding ...troponin C binding / troponin complex / Striated Muscle Contraction / regulation of muscle contraction / cytoskeletal motor activator activity / myosin II complex / sarcomere organization / 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 contraction / skeletal muscle myofibril / actin monomer binding / skeletal muscle fiber development / cardiac muscle contraction / stress fiber / titin binding / actin filament polymerization / filopodium / muscle contraction / actin filament / actin filament organization / 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 / actin cytoskeleton / lamellipodium / actin binding / cell body / hydrolase activity / protein heterodimerization activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / protein homodimerization activity / 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 / らせん対称体再構成法 / 解像度: 35 Å | ||||||
![]() | Wu, S. / Liu, J. / Reedy, M.C. / Tregear, R.T. / Winkler, H. / Franzini-Armstrong, C. / Sasaki, H. / Lucaveche, C. / Goldman, Y.E. / Reedy, M.K. / Taylor, K.A. | ||||||
![]() | ![]() タイトル: Electron tomography of cryofixed, isometrically contracting insect flight muscle reveals novel actin-myosin interactions. 著者: Shenping Wu / Jun Liu / Mary C Reedy / Richard T Tregear / Hanspeter Winkler / Clara Franzini-Armstrong / Hiroyuki Sasaki / Carmen Lucaveche / Yale E Goldman / Michael K Reedy / Kenneth A Taylor / ![]() 要旨: BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of ...BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze ...METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may ...CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments. #1: ジャーナル: J Struct Biol / 年: 2009 タイトル: Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle. 著者: Shenping Wu / Jun Liu / Mary C Reedy / Hanspeter Winkler / Michael K Reedy / Kenneth A Taylor / ![]() 要旨: During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors ...During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different mechanochemical steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze substituted, Araldite embedded, thin-sectioned and stained, to obtain 3D snapshots of the multiplicity of actin-attached myosin structures. We describe procedures for alignment of the repeating lattice of sub-volumes (38.7 nm cross-bridge repeats bounded by troponin) and multivariate data analysis to identify self-similar repeats for computing class averages. Improvements in alignment and classification of repeat sub-volumes reveals (for the first time in active muscle images) the helix of actin subunits in the thin filament and the troponin density with sufficient clarity that a quasiatomic model of the thin filament can be built into the class averages independent of the myosin cross-bridges. We show how quasiatomic model building can identify both strong and weak myosin attachments to actin. We evaluate the accuracy of image classification to enumerate the different types of actin-myosin attachments. | ||||||
履歴 |
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ムービー |
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 1.6 MB | 表示 | ![]() |
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PDB形式 | ![]() | 1.3 MB | 表示 | ![]() |
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-検証レポート
文書・要旨 | ![]() | 586.1 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 930.3 KB | 表示 | |
XML形式データ | ![]() | 206.9 KB | 表示 | |
CIF形式データ | ![]() | 333.9 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
-タンパク質 , 5種, 36分子 0369147Y258ZABCTUVWXDEFGHIJKLM...
#1: タンパク質 | 分子量: 17971.836 Da / 分子数: 4 / 断片: RESIDUES 5-163 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() #2: タンパク質 | 分子量: 10976.568 Da / 分子数: 4 / 断片: RESIDUES 148-237 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() #3: タンパク質 | 分子量: 16304.978 Da / 分子数: 4 / 断片: RESIDUES 4-144 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() #4: タンパク質 | 分子量: 31917.555 Da / 分子数: 8 / 断片: RESIDUES 8-284 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() #5: タンパク質 | 分子量: 41483.117 Da / 分子数: 16 / 断片: RESIDUES 3-374 / 由来タイプ: 天然 / 由来: (天然) ![]() ![]() |
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-非ポリマー , 1種, 16分子 ![](data/chem/img/CA.gif)
#6: 化合物 | ChemComp-CA / |
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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: FILAMENT / 3次元再構成法: らせん対称体再構成法 |
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試料調製
構成要素 | 名称: INSECT FIBRILLAR FLIGHT MUSCLE / タイプ: COMPLEX 詳細: THIS SPECIMEN IS OBTAINED FROM A QUICK FROZEN, ISOMETRICALLY CONTRACTING ASYNCHRONOUS INSECT FLIGHT MUSCLE THAT HAS BEEN FREEZE SUBSTITUTED, PLASTIC EMBEDDED, AND THIN SECTIONED. THE FIBERS ...詳細: THIS SPECIMEN IS OBTAINED FROM A QUICK FROZEN, ISOMETRICALLY CONTRACTING ASYNCHRONOUS INSECT FLIGHT MUSCLE THAT HAS BEEN FREEZE SUBSTITUTED, PLASTIC EMBEDDED, AND THIN SECTIONED. THE FIBERS FOR THIS STUDY WERE SUBJECTED TO MECHANICAL TRANSIENTS. FOR THE QUICK STRETCH, THE FIBER WAS STRETCHED 6 NM PER HALF-SARCOMERE IN 2 MS AND LENGTH WAS HELD CONSTANT AFTER THE LENGTH STEP. THE FREEZING IMPACT OCCURRED 6-7 MS LATER. |
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緩衝液 | 名称: 20 MM MOPS BUFFER, 5 MM NAN3, AND MGCL2, ATP, CACL2, AND EGTA IN VARYING MILLIMOLAR CONCENTRATIONS 詳細: 20 MM MOPS BUFFER, 5 MM NAN3, AND MGCL2, ATP, CACL2, AND EGTA IN VARYING MILLIMOLAR CONCENTRATIONS |
試料 | 包埋: YES / シャドウイング: NO / 染色: NO / 凍結: NO |
試料支持 | 詳細: CARBON |
急速凍結 | 装置: HOMEMADE PLUNGER / 凍結剤: HELIUM 詳細: SMASH AGAINST A LIQUID HELIUM COOLED GOLD COATED COPPER MIRROR |
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電子顕微鏡撮影
顕微鏡 | モデル: FEI/PHILIPS CM300FEG/T / 詳細: NONE |
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電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / Cs: 2 mm |
試料ホルダ | 傾斜角・最大: 72 ° / 傾斜角・最小: -72 ° |
撮影 | フィルム・検出器のモデル: TVIPS TEMCAM-F224 (2k x 2k) |
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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3次元再構成 | 手法: MARKER FREE ALIGNMENT / 解像度の算出法: FSC 0.5 CUT-OFF 詳細: NOTE THAT OUR LOWEST RESOLUTION DATA IS AT INVERSE 1 MICRON. NUMBER OF FOURIER COEFFICIENTS IS ALMOST A HALF MILLION. THESE COORDINATES WERE FIT TO AVERAGED SUBVOLUMES OBTAINED FROM A DUAL ...詳細: NOTE THAT OUR LOWEST RESOLUTION DATA IS AT INVERSE 1 MICRON. NUMBER OF FOURIER COEFFICIENTS IS ALMOST A HALF MILLION. THESE COORDINATES WERE FIT TO AVERAGED SUBVOLUMES OBTAINED FROM A DUAL AXIS TOMOGRAM. THE ACTIN FILAMENT WAS GENERATED TO HAVE THE HELICAL SYMMETRY OF 28 SUBUNITS IN 13 TURNS OF THE 5.9 NM HELIX. THE FITTING WAS DONE MANUALLY USING THE CRYSTALLOGRAPHIC MODEL FITTING PROGRAM O. 対称性のタイプ: HELICAL | ||||||||||||
精密化 | 最高解像度: 35 Å | ||||||||||||
精密化ステップ | サイクル: LAST / 最高解像度: 35 Å
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