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基本情報
登録情報 | データベース: EMDB / ID: EMD-2237 | |||||||||
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タイトル | Electron cyro-microscopy helical reconstruction of Par-3 N-terminal domain | |||||||||
![]() | Reconstruction of Par3NTD | |||||||||
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![]() | cell polarity / DUF3534 domain / self-association | |||||||||
機能・相同性 | ![]() Tight junction interactions / regulation of actin filament-based process / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / internode region of axon / PAR polarity complex / regulation of cellular localization / apical constriction / establishment of centrosome localization / lateral loop / positive regulation of myelination ...Tight junction interactions / regulation of actin filament-based process / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / internode region of axon / PAR polarity complex / regulation of cellular localization / apical constriction / establishment of centrosome localization / lateral loop / positive regulation of myelination / establishment of epithelial cell polarity / Schmidt-Lanterman incisure / bicellular tight junction assembly / myelination in peripheral nervous system / phosphatidylinositol-3-phosphate binding / establishment or maintenance of epithelial cell apical/basal polarity / protein targeting to membrane / wound healing, spreading of cells / centrosome localization / apical junction complex / establishment of cell polarity / negative regulation of peptidyl-threonine phosphorylation / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of receptor internalization / bicellular tight junction / endomembrane system / axonal growth cone / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / adherens junction / protein localization / microtubule cytoskeleton organization / spindle / cell-cell junction / apical part of cell / cell junction / cell cortex / protein phosphatase binding / cell adhesion / cell cycle / apical plasma membrane / cell division / neuronal cell body / protein-containing complex / identical protein binding / 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 / 解像度: 6.1 Å | |||||||||
![]() | Yan Z / Wenjuan W / Jia C / Kai Z / Feng G / Weimin G / Mingjie Z / Fei S / Wei F | |||||||||
![]() | ![]() タイトル: Structural insights into the intrinsic self-assembly of Par-3 N-terminal domain. 著者: Yan Zhang / Wenjuan Wang / Jia Chen / Kai Zhang / Feng Gao / Bingquan Gao / Shuai Zhang / Mingdong Dong / Flemming Besenbacher / Weimin Gong / Mingjie Zhang / Fei Sun / Wei Feng / ![]() 要旨: Par-3, the central organizer of the Par-3/Par-6/atypical protein kinase C complex, is a multimodular scaffold protein that is essential for cell polarity establishment and maintenance. The N-terminal ...Par-3, the central organizer of the Par-3/Par-6/atypical protein kinase C complex, is a multimodular scaffold protein that is essential for cell polarity establishment and maintenance. The N-terminal domain (NTD) of Par-3 is capable of self-association to form filament-like structures, although the underlying mechanism is poorly understood. Here, we determined the crystal structure of Par-3 NTD and solved the filament structure by cryoelectron microscopy. We found that an intrinsic "front-to-back" interaction mode is important for Par-3 NTD self-association and that both the lateral and longitudinal packing within the filament are mediated by electrostatic interactions. Disruptions of the lateral or longitudinal packing significantly impaired Par-3 NTD self-association and thereby impacted the Par-3-mediated epithelial polarization. We finally demonstrated that a Par-3 NTD-like domain from histidine ammonia-lyase also harbors a similar self-association capacity. This work unequivocally provides the structural basis for Par-3 NTD self-association and characterizes one type of protein domain that can self-assemble via electrostatic interactions. | |||||||||
履歴 |
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構造ビューア | EMマップ: ![]() ![]() ![]() |
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画像 | ![]() | 246.1 KB | ||
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-検証レポート
文書・要旨 | ![]() | 273.4 KB | 表示 | ![]() |
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文書・詳細版 | ![]() | 272.5 KB | 表示 | |
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マップ
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注釈 | Reconstruction of Par3NTD | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 0.933 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 : Par-3 N-terminal DUF3534 domain
全体 | 名称: Par-3 N-terminal DUF3534 domain |
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要素 |
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-超分子 #1000: Par-3 N-terminal DUF3534 domain
超分子 | 名称: Par-3 N-terminal DUF3534 domain / タイプ: sample / ID: 1000 / 集合状態: helical filament assembly / Number unique components: 95 |
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分子量 | 理論値: 938 KDa |
-分子 #1: Par-3 N-terminal DUF3534 domain
分子 | 名称: Par-3 N-terminal DUF3534 domain / タイプ: protein_or_peptide / ID: 1 / Name.synonym: Par-3 NTD / コピー数: 95 / 集合状態: helical filament assembly / 組換発現: Yes |
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由来(天然) | 生物種: ![]() ![]() |
分子量 | 理論値: 938 KDa |
組換発現 | 生物種: ![]() ![]() |
-実験情報
-構造解析
手法 | 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
濃度 | 2.0 mg/mL |
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緩衝液 | pH: 8 / 詳細: 50 mM Tris, 100 mM NaCl, 1 mM DTT and 1 mM EDTA |
グリッド | 詳細: 300-mesh GiGTM holy carbon grid |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV / 手法: blotted 3.0 s with a blot force 3 |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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温度 | 平均: 85 K |
アライメント法 | Legacy - 非点収差: objective lens astigmatism was corrected at 96,000 times magnification Legacy - Electron beam tilt params: 0 |
日付 | 2010年12月1日 |
撮影 | カテゴリ: CCD フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k) 実像数: 6460 / 平均電子線量: 20 e/Å2 詳細: The images were automatically collected by using leginon system. ビット/ピクセル: 32 |
Tilt angle min | 0 |
Tilt angle max | 0 |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): -2.5 µm / 最小 デフォーカス(公称値): -1.8 µm / 倍率(公称値): 96000 |
試料ステージ | 試料ホルダー: Liquid nitrogen cooled 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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画像解析
詳細 | The initial model was obtained IHRSR. Then particles were aligned using EMAN1. |
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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: 3.53 Å 想定した対称性 - らせんパラメータ - ΔΦ: 43.84 ° 想定した対称性 - らせんパラメータ - 軸対称性: C1 (非対称) アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 6.1 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: Spider, EMAN1 詳細: The resolution was assessed by splitting original particles set into two halves and comparing two independent reconstructions. The final map was reconstructed by using the whole set. |
CTF補正 | 詳細: each image |
最終 角度割当 | 詳細: Euler angle system in EMAN1 |
-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 |
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ソフトウェア | 名称: Chimera and NAMD2 |
詳細 | Protocol: Rigid body fitting first and then molecular dynamics flexible fitting. Crystal structure of the monomer was docked as a rigid body into the cryoEM map using UCSF Chimera and applied with the helical symmetry to build the initial model. Then the structural model was solvated in a box of water molecules with 150 mM NaCl in VMD, using 15 angstrom of padding in all directions. Extra ions were added to neutralize the systems. The simulations were performed with program NAMD 2.8, using the CHARMM27 force field with CMAP corrections. All the fitting procedure is the same as the application of symmetry-restrained MDFF to chaperonin reported previously. |
精密化 | 空間: REAL / プロトコル: FLEXIBLE FIT 当てはまり具合の基準: cross correlation coefficient |
得られたモデル | ![]() PDB-3zee: |