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基本情報
登録情報 | データベース: PDB / ID: 6t1r | |||||||||
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タイトル | Pseudo-atomic model of a 16-mer assembly of reduced recombinant human alphaA-crystallin (non domain swapped configuration) | |||||||||
![]() | Alpha-crystallin A chain | |||||||||
![]() | CHAPERONE / sHsp / alphaA-crystallin / domain swapping | |||||||||
機能・相同性 | ![]() negative regulation of intracellular transport / response to stimulus / structural constituent of eye lens / visual perception / unfolded protein binding / protein stabilization / negative regulation of apoptotic process / structural molecule activity / protein-containing complex / nucleoplasm ...negative regulation of intracellular transport / response to stimulus / structural constituent of eye lens / visual perception / unfolded protein binding / protein stabilization / negative regulation of apoptotic process / structural molecule activity / protein-containing complex / nucleoplasm / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm 類似検索 - 分子機能 | |||||||||
生物種 | ![]() | |||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 9.8 Å | |||||||||
![]() | Peters, C. / Kaiser, C.J.O. / Weinkauf, S. / Zacharias, M. / Buchner, J. | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: The structure and oxidation of the eye lens chaperone αA-crystallin. 著者: Christoph J O Kaiser / Carsten Peters / Philipp W N Schmid / Maria Stavropoulou / Juan Zou / Vinay Dahiya / Evgeny V Mymrikov / Beate Rockel / Sam Asami / Martin Haslbeck / Juri Rappsilber / ...著者: Christoph J O Kaiser / Carsten Peters / Philipp W N Schmid / Maria Stavropoulou / Juan Zou / Vinay Dahiya / Evgeny V Mymrikov / Beate Rockel / Sam Asami / Martin Haslbeck / Juri Rappsilber / Bernd Reif / Martin Zacharias / Johannes Buchner / Sevil Weinkauf / ![]() ![]() 要旨: The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the ...The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens. | |||||||||
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 375.8 KB | 表示 | ![]() |
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PDB形式 | ![]() | 258.5 KB | 表示 | ![]() |
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その他 | ![]() |
-検証レポート
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: 19936.314 Da / 分子数: 16 / 由来タイプ: 組換発現 / 詳細: wild type residues 1-166 / 由来: (組換発現) ![]() ![]() ![]() |
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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: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Reduced recombinant human alphaA-crystallin / タイプ: COMPLEX 詳細: Recombinant wild type full-length human alphaA-crystallin purified in the presence of reductant. Entity ID: all / 由来: RECOMBINANT | |||||||||||||||||||||||||||||||||||
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分子量 | 値: 0.319 MDa / 実験値: NO | |||||||||||||||||||||||||||||||||||
由来(天然) | 生物種: ![]() | |||||||||||||||||||||||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | |||||||||||||||||||||||||||||||||||
緩衝液 | pH: 7.4 詳細: Buffer was prepared without EDTA and DTT. EDTA stock (500mM) was titrated to pH 8 and added to 1 mM. DTT stock (1M) was added to 1mM. | |||||||||||||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 0.3 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: Specimen was thawed, diluted to the final concentration and equilibrated at 310K for 3h. | |||||||||||||||||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 200 divisions/in. / グリッドのタイプ: Quantifoil R2/1 | |||||||||||||||||||||||||||||||||||
急速凍結 | 装置: HOMEMADE PLUNGER / 凍結剤: ETHANE / 凍結前の試料温度: 293 K 詳細: Diluted equilibrated specimen was added to glow-discharged (30s) grids. Sample was blotted 30s after sample application and immediately plunged. |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 37000 X / 倍率(補正後): 37037 X / 最大 デフォーカス(公称値): 2500 nm / 最小 デフォーカス(公称値): 1200 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm / アライメント法: COMA FREE |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 平均露光時間: 3.2 sec. / 電子線照射量: 30 e/Å2 / 検出モード: SUPER-RESOLUTION フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 撮影したグリッド数: 3 |
電子光学装置 | エネルギーフィルタースリット幅: 10 eV |
画像スキャン | 動画フレーム数/画像: 10 / 利用したフレーム数/画像: 1-10 |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING ONLY | ||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 74068 / 詳細: particles were picked manually | ||||||||||||||||||||||||||||
対称性 | 点対称性: D4 (2回x4回 2面回転対称) | ||||||||||||||||||||||||||||
3次元再構成 | 解像度: 9.8 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 19783 / 対称性のタイプ: POINT | ||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT 詳細: Structure created by homology modelling using PDB 3N3E and the N-terminal region (1-60) modelled using I-Tasser. The model was refined by flexible fitting into the EM density map EMD-4894. ...詳細: Structure created by homology modelling using PDB 3N3E and the N-terminal region (1-60) modelled using I-Tasser. The model was refined by flexible fitting into the EM density map EMD-4894. The N-terminus is modelled only as CA atoms. | ||||||||||||||||||||||||||||
原子モデル構築 | PDB-ID: 3N3E Accession code: 3N3E / Source name: PDB / タイプ: experimental model |