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基本情報

登録情報
データベース: PDB / ID: 3jax
タイトルHeavy meromyosin from Schistosoma mansoni muscle thick filament by negative stain EM
要素
  • myosin 2 heavy chain
  • myosin regulatory light chain
  • smooth muscle myosin essential light chain
キーワードCONTRACTILE PROTEIN / muscle protein / smooth muscle / myosin subfragment 2 / heavy meromyosin / essential light chain / regulatory light chain / motor protein / coiled-coil
機能・相同性
機能・相同性情報


myosin complex / cytoskeletal motor activity / actin filament binding / calcium ion binding / ATP binding
類似検索 - 分子機能
: / EF-hand domain / Myosin N-terminal SH3-like domain / IQ calmodulin-binding motif / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head, motor domain ...: / EF-hand domain / Myosin N-terminal SH3-like domain / IQ calmodulin-binding motif / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / IQ motif, EF-hand binding site / Kinesin motor domain superfamily / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
類似検索 - ドメイン・相同性
Myosin 2 heavy chain / Smooth muscle myosin essential light chain / Myosin regulatory light chain
類似検索 - 構成要素
生物種Schistosoma mansoni (マンソン住血吸虫)
手法電子顕微鏡法 / らせん対称体再構成法 / ネガティブ染色法 / 解像度: 23 Å
データ登録者Sulbaran, G. / Alamo, L. / Pinto, A. / Marquez, G. / Mendez, F. / Padron, R. / Craig, R.
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2015
タイトル: An invertebrate smooth muscle with striated muscle myosin filaments.
著者: Guidenn Sulbarán / Lorenzo Alamo / Antonio Pinto / Gustavo Márquez / Franklin Méndez / Raúl Padrón / Roger Craig /
要旨: Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and ...Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components.
#1: ジャーナル: J Mol Biol / 年: 2008
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.
#2: ジャーナル: J Mol Biol / 年: 2003
タイトル: Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction.
著者: Jun Liu / Thomas Wendt / Dianne Taylor / Kenneth Taylor /
要旨: The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact ...The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.
#3: ジャーナル: Proc Natl Acad Sci U S A / 年: 2006
タイトル: Crystal structures of human cardiac beta-myosin II S2-Delta provide insight into the functional role of the S2 subfragment.
著者: Wulf Blankenfeldt / Nicolas H Thomä / John S Wray / Mathias Gautel / Ilme Schlichting /
要旨: Myosin II is the major component of the muscle thick filament. It consists of two N-terminal S1 subfragments ("heads") connected to a long dimeric coiled-coil rod. The rod is in itself twofold ...Myosin II is the major component of the muscle thick filament. It consists of two N-terminal S1 subfragments ("heads") connected to a long dimeric coiled-coil rod. The rod is in itself twofold symmetric, but in the filament, the two heads point away from the filament surface and are therefore not equivalent. This breaking of symmetry requires the initial section of the rod, subfragment 2 (S2), to be relatively flexible. S2 is an important functional element, involved in various mechanisms by which the activity of smooth and striated muscle is regulated. We have determined crystal structures of the 126 N-terminal residues of S2 from human cardiac beta-myosin II (S2-Delta), of both WT and the disease-associated E924K mutant. S2-Delta is a straight parallel dimeric coiled coil, but the N terminus of one chain is disordered in WT-S2-Delta due to crystal contacts, indicative of unstable local structure. Bulky noncanonical side chains pack into a/d positions of S2-Delta's N terminus, leading to defined local asymmetry and axial stagger, which could induce nonequivalence of the S1 subfragments. Additionally, S2 possesses a conserved charge distribution with three prominent rings of negative potential within S2-Delta, the first of which may provide a binding interface for the "blocked head" of smooth muscle myosin in the OFF state. The observation that many disease-associated mutations affect the second negatively charged ring further suggests that charge interactions play an important role in regulation of cardiac muscle activity through myosin-binding protein C.
#4: ジャーナル: Cell / 年: 1999
タイトル: Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head.
著者: A Houdusse / V N Kalabokis / D Himmel / A G Szent-Györgyi / C Cohen /
要旨: The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin ...The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.
履歴
登録2015年7月3日登録サイト: RCSB / 処理サイト: RCSB
改定 1.02015年10月7日Provider: repository / タイプ: Initial release
改定 1.12015年10月21日Group: Database references
改定 1.22015年11月4日Group: Database references
改定 1.32018年7月18日Group: Author supporting evidence / Data collection / カテゴリ: em_single_particle_entity / em_software / Item: _em_software.image_processing_id

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構造の表示

ムービー
  • 生物学的単位 - representative helical assembly
  • Jmolによる作画
  • ダウンロード
  • 登録構造単位
  • Jmolによる作画
  • ダウンロード
  • 単純化した表面モデル + あてはめた原子モデル
  • マップデータ: EMDB-6370
  • Jmolによる作画
  • ダウンロード
  • EMマップとの重ね合わせ
  • マップデータ: EMDB-6370
  • UCSF Chimeraによる作画
  • ダウンロード
ムービービューア
構造ビューア分子:
MolmilJmol/JSmol

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集合体

登録構造単位
A: myosin 2 heavy chain
B: myosin 2 heavy chain
C: smooth muscle myosin essential light chain
D: smooth muscle myosin essential light chain
E: myosin regulatory light chain
F: myosin regulatory light chain


分子量 (理論値)分子数
合計 (水以外)301,7926
ポリマ－301,7926
非ポリマー00
水00
1
A: myosin 2 heavy chain
B: myosin 2 heavy chain
C: smooth muscle myosin essential light chain
D: smooth muscle myosin essential light chain
E: myosin regulatory light chain
F: myosin regulatory light chain
x 20


分子量 (理論値)分子数
合計 (水以外)6,035,839120
ポリマ－6,035,839120
非ポリマー00
水0
タイプ名称対称操作数
helical symmetry operation20
2


  • 登録構造と同一（異なる座標系）
  • helical asymmetric unit
タイプ名称対称操作数
helical symmetry operation1
3


  • 登録構造と同一（異なる座標系）
  • helical asymmetric unit, std helical frame
タイプ名称対称操作数
transform to helical frame1
対称性らせん対称: (回転対称性: 4 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 20 / Rise per n subunits: 145 Å / Rotation per n subunits: 30 °)

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要素

#1: タンパク質 myosin 2 heavy chain


分子量: 112182.516 Da / 分子数: 2 / 由来タイプ: 天然
由来: (天然) Schistosoma mansoni (マンソン住血吸虫)
細胞内の位置: sarcomere / Organelle: myosin thick filament / 株: JL / 組織: smooth muscle / 参照: UniProt: A0A0R4I956*PLUS
#2: タンパク質 smooth muscle myosin essential light chain


分子量: 17004.221 Da / 分子数: 2 / 由来タイプ: 天然
由来: (天然) Schistosoma mansoni (マンソン住血吸虫)
細胞内の位置: sarcomere / Organelle: myosin thick filament / 株: JL / 組織: smooth muscle / 参照: UniProt: A0A0R4I957*PLUS
#3: タンパク質 myosin regulatory light chain


分子量: 21709.250 Da / 分子数: 2 / 由来タイプ: 天然
由来: (天然) Schistosoma mansoni (マンソン住血吸虫)
細胞内の位置: sarcomere / Organelle: myosin thick filament / 株: JL / 組織: smooth muscle / 参照: UniProt: A0A0R4I958*PLUS
配列の詳細THE IMAGED FILAMENTS ARE FROM SCHISTOSOMA MANSONI, BUT THE MODELED SEQUENCES ARE FROM CHICKEN/HUMAN ...THE IMAGED FILAMENTS ARE FROM SCHISTOSOMA MANSONI, BUT THE MODELED SEQUENCES ARE FROM CHICKEN/HUMAN CHIMERA (CHAINS A,B), CHICKEN (CHAINS C,D), AND TARANTULA (CHAINS E,F). SEQUENCE CONFLICTS FOR CHAINS A AND B ARE CONSISTENT WITH PDB ENTRIES 1BR1 AND 1I84. THE SEQUENCE OF THE HEAVY CHAIN STRUCTURE REPORTED HERE DIFFERS FROM THAT REPORTED IN UNP P10587. SER 2 TO ALA IS A CLONING ARTIFACT IN PDB ENTRY 1BR1. PEPTIDE CHAIN DESIGNATIONS: THE TERMS "BLOCKED" AND "FREE" REFER TO THE CONFORMATIONS OF THE TWO S1 MYOSIN HEADS. "FREE" MYOSIN HEAD MYOSIN HEAVY CHAIN S1 PLUS S2 FRAGMENT IS CHAIN A ELC IS CHAIN C RLC IS CHAIN E "BLOCKED" MYOSIN HEAD MYOSIN HEAVY CHAIN S1 PLUS S2 FRAGMENT IS CHAIN B ELC IS CHAIN D RLC IS CHAIN F SEQUENCE GAPS IN THE MOLECULAR MODEL: HEAVY CHAIN UNP P10587 CHAINS A AND B: 1, 205-210, 452-457, 635-655, 853-1979 HEAVY CHAIN S2 FRAGMENT UNP P12883 CHAIN A: 1-841, 962-1935 HEAVY CHAIN S2 FRAGMENT UNP P12883 CHAIN B: 1-841, 964-1935 ELC UNP P02607 CHAINS C AND D: 1-3

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実験情報

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実験

実験手法: 電子顕微鏡法
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
ID名称タイプ詳細Parent-ID
1Myosin thick filaments from Schistosoma mansoni smooth muscleCOMPLEXPolymer of myosin II molecules helically assembled over a paramyosin core0
2Myosin II1
緩衝液名称: 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM PIPES, 1mM NaN3, 5 mM MgATP, 0.01 mM blebbistatin, protease inhibitor cocktail (Sigma P-8465)
pH: 7
詳細: 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM PIPES, 1mM NaN3, 5 mM MgATP, 0.01 mM blebbistatin, protease inhibitor cocktail (Sigma P-8465)
試料包埋: NO / シャドウイング: NO / 染色: YES / 凍結: NO
詳細: One drop of filament suspension was placed on grids and negatively stained with 1% uranyl acetate.
染色タイプ: NEGATIVE / 染色剤: Uranyl Acetate
試料支持詳細: 400-mesh holey carbon grids. Specimens were imaged on thin carbon extending over the holes.

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電子顕微鏡撮影

顕微鏡モデル: FEI/PHILIPS CM120T / 日付: 2013年2月15日 / 詳細: 1.5 post-magnification, low-dose conditions
電子銃電子線源: LAB6 / 加速電圧: 80 kV / 照射モード: FLOOD BEAM
電子レンズモード: BRIGHT FIELD / 倍率（公称値）: 42000 X / 倍率（補正後）: 42000 X / 最大 デフォーカス（公称値）: 2400 nm / 最小 デフォーカス（公称値）: 600 nm / Cs: 2 mm
非点収差: Objective lens astigmatism was corrected at 240,000 times magnification
試料ホルダ試料ホルダーモデル: SIDE ENTRY, EUCENTRIC / 資料ホルダタイプ: Room temperature holder
撮影電子線照射量: 10 e/Å2
フィルム・検出器のモデル: TVIPS TEMCAM-F224 (2k x 2k)
画像スキャンデジタル画像の数: 263
放射プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray
放射波長相対比: 1

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解析

EMソフトウェア
ID名称バージョンカテゴリ
1UCSF Chimeraモデルフィッティング
2EMAN2３次元再構成
3SPIDER３次元再構成
らせん対称回転角度/サブユニット: 30 ° / 軸方向距離/サブユニット: 145 Å / らせん対称軸の対称性: C4
詳細: Heavy meromyosin (HMM) is the outermost helical unit of the myosin thick filament formed by two myosin heads that include the motor domains, the proteolytic fragment of myosin heavy chain ...詳細: Heavy meromyosin (HMM) is the outermost helical unit of the myosin thick filament formed by two myosin heads that include the motor domains, the proteolytic fragment of myosin heavy chain subfragment 2 (S2), and two associated light chains (ELC and RLC).
3次元再構成手法: Single particle reconstruction with a modification of the IHRSR method
解像度: 23 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 9500 / ピクセルサイズ（公称値）: 5.7 Å / ピクセルサイズ（実測値）: 5.7 Å
詳細: For each iteration of reconstruction (30 cycles), filament segment projections were compared with different projections of the reference reconstruction as follows: seven 2.3 nm axial shifts, ...詳細: For each iteration of reconstruction (30 cycles), filament segment projections were compared with different projections of the reference reconstruction as follows: seven 2.3 nm axial shifts, 2 degree intervals of rotation about the filament axis up to 90 degrees, and 2 degree intervals of out-of-plane tilting from -10 degrees to +10 degrees. The total number of projections was 7 x 45 x 11 = 3465. For the final 19 cycles of the reconstruction, we used only the best-ordered 420 filament halves (those in which >30% of the segments were found good enough to be used by the reconstruction script in the back-projection in previous cycles). From ~17,000 segments, ~9,500 (56%) were included in the final reconstruction. This final 3D-reconstruction was the average of the last 19 reconstructions between cycles 12 - 30. Its resolution, according to the 0.5 Fourier Shell Correlation (FSC) criterion, was 2.3 nm.
対称性のタイプ: HELICAL
原子モデル構築プロトコル: RIGID BODY FIT / 空間: REAL
詳細: METHOD--rigid docking REFINEMENT PROTOCOL--RIGID BODY DETAILS--3DTP was fitted as a rigid body using the Fit in Map tool of UCSF Chimera.
原子モデル構築
IDPDB-IDPDB chain-ID 3D fitting-ID
13DTP

3dtp

A1
23DTP

3dtp

B1
33DTP

3dtp

C1
43DTP

3dtp

D1
53DTP

3dtp

E1
63DTP

3dtp

F1
精密化ステップサイクル: LAST
タンパク質核酸リガンド溶媒全体
原子数20580 0 0 0 20580

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