ジャーナル: Cell / 年: 2007 タイトル: Structure of full-length HIV-1 CA: a model for the mature capsid lattice. 著者: Barbie K Ganser-Pornillos / Anchi Cheng / Mark Yeager / 要旨: The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids are modeled as fullerene structures composed of closed ...The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids are modeled as fullerene structures composed of closed hexameric arrays of the viral CA protein, but a high-resolution structure of the lattice has remained elusive. A three-dimensional map of two-dimensional crystals of the R18L mutant of HIV-1 CA was derived by electron cryocrystallography. The docking of high-resolution domain structures into the map yielded the first unambiguous model for full-length HIV-1 CA. Three important protein-protein assembly interfaces are required for capsid formation. Each CA hexamer is composed of an inner ring of six N-terminal domains and an outer ring of C-terminal domains that form dimeric linkers connecting neighboring hexamers. Interactions between the two domains of CA further stabilize the hexamer and provide a structural explanation for the mechanism of action of known HIV-1 assembly inhibitors.
濃度: 32 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: Carbon coated molybdenum grids
急速凍結
凍結剤: ETHANE 詳細: Washed with 0.1M KCl, blotted briefly, and plunged into liquid ethane
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データ収集
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
顕微鏡
モデル: FEI TECNAI F20
電子銃
電子線源: FIELD EMISSION GUN / 加速電圧: 120 kV / 照射モード: OTHER
電子レンズ
モード: BRIGHT FIELD / 倍率(公称値): 52000 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): 400 nm / Cs: 2 mm
試料ホルダ
温度: 90 K / 傾斜角・最大: 40.3 ° / 傾斜角・最小: 0 °
撮影
電子線照射量: 10 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM
放射
プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: electron
放射波長
相対比: 1
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解析
EMソフトウェア
ID
名称
カテゴリ
1
SITUS COLACOR
モデルフィッティング
2
SITUS COLORES
モデルフィッティング
3
MRC
3次元再構成
3次元再構成
手法: 2D crystallography / 解像度: 9 Å 詳細: THIS MODDEL CONTAINS CA ATOMS ONLY. THE TWO DOMAINS UNP RESIDUES 133-280 AND UNP RESIDUES 281-351 WERE ALLOWED TO MOVE INDEPENDENTLY. THAT IS WHY THE CONNECTIVITY BETWEEN THEM IS NOT WELL- ...詳細: THIS MODDEL CONTAINS CA ATOMS ONLY. THE TWO DOMAINS UNP RESIDUES 133-280 AND UNP RESIDUES 281-351 WERE ALLOWED TO MOVE INDEPENDENTLY. THAT IS WHY THE CONNECTIVITY BETWEEN THEM IS NOT WELL-PRESERVED. MRC AND CCP4 PROGRAMS WERE USED. 対称性のタイプ: 2D CRYSTAL
原子モデル構築
プロトコル: RIGID BODY FIT / 空間: REAL / 詳細: REFINEMENT PROTOCOL--RIGID BODY