ジャーナル: Proc Natl Acad Sci U S A / 年: 2011 タイトル: Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach. 著者: Nathalie Braun / Martin Zacharias / Jirka Peschek / Andreas Kastenmüller / Juan Zou / Marianne Hanzlik / Martin Haslbeck / Juri Rappsilber / Johannes Buchner / Sevil Weinkauf / 要旨: The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it ...The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.
SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AC" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AC" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 18-STRANDED BARREL THIS IS REPRESENTED BY A 19-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "GC" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 18-STRANDED BARREL THIS IS REPRESENTED BY A 19-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "MC" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 18-STRANDED BARREL THIS IS REPRESENTED BY A 19-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "SC" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 18-STRANDED BARREL THIS IS REPRESENTED BY A 19-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.
A: ALPHA-CRYSTALLIN B CHAIN B: ALPHA-CRYSTALLIN B CHAIN C: ALPHA-CRYSTALLIN B CHAIN D: ALPHA-CRYSTALLIN B CHAIN E: ALPHA-CRYSTALLIN B CHAIN F: ALPHA-CRYSTALLIN B CHAIN G: ALPHA-CRYSTALLIN B CHAIN H: ALPHA-CRYSTALLIN B CHAIN I: ALPHA-CRYSTALLIN B CHAIN J: ALPHA-CRYSTALLIN B CHAIN K: ALPHA-CRYSTALLIN B CHAIN L: ALPHA-CRYSTALLIN B CHAIN M: ALPHA-CRYSTALLIN B CHAIN N: ALPHA-CRYSTALLIN B CHAIN O: ALPHA-CRYSTALLIN B CHAIN P: ALPHA-CRYSTALLIN B CHAIN Q: ALPHA-CRYSTALLIN B CHAIN R: ALPHA-CRYSTALLIN B CHAIN S: ALPHA-CRYSTALLIN B CHAIN T: ALPHA-CRYSTALLIN B CHAIN U: ALPHA-CRYSTALLIN B CHAIN V: ALPHA-CRYSTALLIN B CHAIN W: ALPHA-CRYSTALLIN B CHAIN X: ALPHA-CRYSTALLIN B CHAIN
濃度: 0.2 mg/ml / 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
急速凍結
凍結剤: ETHANE 詳細: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 50, METHOD- BLOT FOR 1 SECOND BEFORE PLUNGING,
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電子顕微鏡撮影
顕微鏡
モデル: JEOL 2010HT
電子銃
電子線源: LAB6 / 加速電圧: 120 kV / 照射モード: SPOT SCAN
電子レンズ
モード: BRIGHT FIELD / 倍率(公称値): 50000 X / 倍率(補正後): 47000 X / 最大 デフォーカス(公称値): 1500 nm / 最小 デフォーカス(公称値): 600 nm
試料ホルダ
温度: 100 K
撮影
電子線照射量: 10 e/Å2
画像スキャン
デジタル画像の数: 33
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解析
EMソフトウェア
名称: IMAGIC / カテゴリ: 3次元再構成
CTF補正
詳細: EACH MICROGRAPH, PHASE FLIPPING
対称性
点対称性: C1 (非対称)
3次元再構成
手法: PROJECTION MATCHING / 解像度: 9.4 Å / 粒子像の数: 17560 / ピクセルサイズ(公称値): 1.69 Å / ピクセルサイズ(実測値): 1.8 Å 詳細: SUBMISSION BASED ON EXPERIMENTAL DATA EMDB EMD-1894.(DEPOSITION ID: 7925). 対称性のタイプ: POINT