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- PDB-3jbh: TWO HEAVY MEROMYOSIN INTERACTING-HEADS MOTIFS FLEXIBLE DOCKED INT... -

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基本情報

登録情報
データベース: PDB / ID: 3jbh
タイトルTWO HEAVY MEROMYOSIN INTERACTING-HEADS MOTIFS FLEXIBLE DOCKED INTO TARANTULA THICK FILAMENT 3D-MAP ALLOWS IN DEPTH STUDY OF INTRA- AND INTERMOLECULAR INTERACTIONS
要素
  • MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
  • MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
  • MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
キーワードCONTRACTILE PROTEIN / MYOSIN INTERACTING-HEADS MOTIF / CRYO-EM (低温電子顕微鏡法) / THICK FILAMENT (ミオフィラメント) / FLEXIBLE DOCKING / SINGLE PARTICLE RECONSTRUCTION (単粒子解析法) / ITERATIVE HELICAL REAL SPACE RECONSTRUCTION (IHRSR) / INTER- AND INTRAMOLECULAR INTERACTIONS / MYOSIN REGULATION / SUPER-RELAXATION / STRIATED MUSCLE (横紋筋) / TARANTULA (オオツチグモ科)
機能・相同性
機能・相同性情報


myosin filament / myosin complex / myofibril / cytoskeletal motor activity / actin filament binding / calcium ion binding / ATP binding
類似検索 - 分子機能
Myosin light chain Mlc1/alkali / : / EF-hand domain / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. ...Myosin light chain Mlc1/alkali / : / EF-hand domain / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / EF-hand domain pair / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / 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 striated muscle / Myosin 2 essential light chain striated muscle / Myosin 2 regulatory light chain striated muscle
類似検索 - 構成要素
生物種Aphonopelma (クモ)
手法電子顕微鏡法 / らせん対称体再構成法 / クライオ電子顕微鏡法 / 解像度: 20 Å
データ登録者Alamo, L. / Qi, D. / Wriggers, W. / Pinto, A. / Zhu, J. / Bilbao, A. / Gillilan, R.E. / Hu, S. / Padron, R.
引用
ジャーナル: J Mol Biol / 年: 2016
タイトル: Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.
著者: Lorenzo Alamo / Dan Qi / Willy Wriggers / Antonio Pinto / Jingui Zhu / Aivett Bilbao / Richard E Gillilan / Songnian Hu / Raúl Padrón /
要旨: Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and ...Tarantula striated muscle is an outstanding system for understanding the molecular organization of myosin filaments. Three-dimensional reconstruction based on cryo-electron microscopy images and single-particle image processing revealed that, in a relaxed state, myosin molecules undergo intramolecular head-head interactions, explaining why head activity switches off. The filament model obtained by rigidly docking a chicken smooth muscle myosin structure to the reconstruction was improved by flexibly fitting an atomic model built by mixing structures from different species to a tilt-corrected 2-nm three-dimensional map of frozen-hydrated tarantula thick filament. We used heavy and light chain sequences from tarantula myosin to build a single-species homology model of two heavy meromyosin interacting-heads motifs (IHMs). The flexibly fitted model includes previously missing loops and shows five intramolecular and five intermolecular interactions that keep the IHM in a compact off structure, forming four helical tracks of IHMs around the backbone. The residues involved in these interactions are oppositely charged, and their sequence conservation suggests that IHM is present across animal species. The new model, PDB 3JBH, explains the structural origin of the ATP turnover rates detected in relaxed tarantula muscle by ascribing the very slow rate to docked unphosphorylated heads, the slow rate to phosphorylated docked heads, and the fast rate to phosphorylated undocked heads. The conservation of intramolecular interactions across animal species and the presence of IHM in bilaterians suggest that a super-relaxed state should be maintained, as it plays a role in saving ATP in skeletal, cardiac, and smooth muscles.
#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: BMC Genomics / 年: 2009
タイトル: Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms.
著者: Jingui Zhu / Yongqiao Sun / Fa-Qing Zhao / Jun Yu / Roger Craig / Songnian Hu /
要旨: BACKGROUND: Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking.
RESULTS: We constructed a cDNA library from Aphonopelma sp. (Tarantula) skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags) from randomly picked clones. EST analysis showed 305 ...RESULTS: We constructed a cDNA library from Aphonopelma sp. (Tarantula) skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags) from randomly picked clones. EST analysis showed 305 unigenes, among which 81 had more than 2 ESTs. Twenty abundant unigenes had matches to skeletal muscle-related genes including actin, myosin, tropomyosin, troponin-I, T and C, paramyosin, muscle LIM protein, muscle protein 20, a-actinin and tandem Ig/Fn motifs (found in giant sarcomere-related proteins). Matches to myosin light chain kinase and calponin were also identified. These results support the existence of both actin-linked and myosin-linked regulation in tarantula skeletal muscle. We have predicted full-length as well as partial cDNA sequences both experimentally and computationally for myosin heavy and light chains, actin, tropomyosin, and troponin-I, T and C, and have deduced the putative peptides. A preliminary analysis of the structural and functional properties was also carried out. Sequence similarities suggested multiple isoforms of most myofibrillar proteins, supporting the generality of multiple isoforms known from previous muscle sequence studies. This may be related to a mix of muscle fiber types.
CONCLUSION: The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and ...CONCLUSION: The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and functional observations in this model species.
履歴
登録2015年9月1日登録サイト: RCSB / 処理サイト: RCSB
改定 1.02016年3月9日Provider: repository / タイプ: Initial release
改定 1.12016年12月14日Group: Database references
改定 1.22018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
改定 1.32024年2月21日Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
カテゴリ: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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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-1950
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  • ダウンロード
  • EMマップとの重ね合わせ
  • マップデータ: EMDB-1950
  • UCSF Chimera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-1950
  • 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 STRIATED MUSCLE
B: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
C: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
D: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
E: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
F: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
G: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
H: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
I: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
J: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
K: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
L: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE


分子量 (理論値)分子数
合計 (水以外)1,055,85912
ポリマ－1,055,85912
非ポリマー00
水0
1
A: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
B: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
C: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
D: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
E: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
F: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
G: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
H: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
I: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
J: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
K: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
L: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
x 20


分子量 (理論値)分子数
合計 (水以外)21,117,173240
ポリマ－21,117,173240
非ポリマー00
水0
タイプ名称対称操作数
helical symmetry operation19
identity operation1_555x,y,z1
2


  • 登録構造と同一
  • helical asymmetric unit
タイプ名称対称操作数
identity operation1_555x,y,z1
3


  • 登録構造と同一
  • helical asymmetric unit, std helical frame
タイプ名称対称操作数
identity operation1_555x,y,z1
対称性らせん対称: (回転対称性: 4 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 1 / Rise per n subunits: 100 Å / Rotation per n subunits: 30 °)

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要素

#1: タンパク質
MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE


分子量: 224542.281 Da / 分子数: 4 / 断片: SUBFRAGMENT 1 (S1) AND SUBFRAGMENT 2 (S2) / 由来タイプ: 天然 / 由来: (天然) Aphonopelma (クモ) / 組織: LEG MUSCLE / 参照: UniProt: A0A140UGH3*PLUS
#2: タンパク質
MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE


分子量: 17628.104 Da / 分子数: 4 / 断片: ESSENTIAL LIGHT CHAIN (ELC) / 由来タイプ: 天然 / 由来: (天然) Aphonopelma (クモ) / 組織: LEG MUSCLE / 参照: UniProt: A0A140UGH4*PLUS
#3: タンパク質
MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE


分子量: 21794.281 Da / 分子数: 4 / 断片: REGULATORY LIGHT CHAIN (RLC) / 由来タイプ: 天然 / 由来: (天然) Aphonopelma (クモ) / 組織: LEG MUSCLE / 参照: UniProt: A0A140UGH5*PLUS

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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 filaments from Tarantula striated muscleCOMPLEXPolymer of a multiple myosin assembled over a paramyosin core0
2MYOSIN IIミオシン1
緩衝液名称: 100mM NaCl, 3mM MgCl2, 1mM EGTA, 5mM PIPES, 5mM NaH2PO4, 1mM NaN3
pH: 7
詳細: 100mM NaCl, 3mM MgCl2, 1mM EGTA, 5mM PIPES, 5mM NaH2PO4, 1mM NaN3
試料包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
試料支持詳細: HOLEY CARBON GRIDS.
急速凍結装置: HOMEMADE PLUNGER / 凍結剤: ETHANE / Temp: 93 K / 湿度: 80 %
詳細: 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 ...詳細: 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: 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 ...手法: 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.

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電子顕微鏡撮影

顕微鏡モデル: FEI/PHILIPS CM120T / 日付: 2002年10月23日 / 詳細: low dose
電子銃電子線源: LAB6 / 加速電圧: 120 kV / 照射モード: FLOOD BEAM
電子レンズモード: BRIGHT FIELDBright-field microscopy / 倍率（公称値）: 35000 X / 倍率（補正後）: 35000 X / 最大 デフォーカス（公称値）: 1950 nm / 最小 デフォーカス（公称値）: 1950 nm / Cs: 2 mm
試料ホルダ温度: 88 K / 最高温度: 90 K / 最低温度: 88 K
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: 1008
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
1Situs2.3モデルフィッティング
2SPIDER３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: 100 Å / らせん対称軸の対称性: C4
3次元再構成手法: Single particle reconstruction with a modification of the IHRSR method
解像度: 20 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ピクセルサイズ（公称値）: 2.48 Å / ピクセルサイズ（実測値）: 2.48 Å
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. 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.
対称性のタイプ: HELICAL
原子モデル構築プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: Correlation
詳細: METHOD--FLEXIBLE FITTING DETAILS--Protocol- Flexible Fitting. The flexible docking procedure is based on a connected (motion capture) network of identified features within the atomic model. ...詳細: METHOD--FLEXIBLE FITTING DETAILS--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
原子モデル構築PDB-ID: 3DTP

3dtp


Accession code: 3DTP / Source name: PDB / タイプ: experimental model
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41968 0 0 0 41968

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