ジャーナル: Nat Struct Biol / 年: 2003 タイトル: Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A. 著者: Xing Zhang / Stephen B Walker / Paul R Chipman / Max L Nibert / Timothy S Baker / 要旨: Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, ...Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 A by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase lambda 3, were then fitted into the density map. Each copy of lambda 3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein lambda 1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of lambda 3 is bound per vertex. lambda 3 is furthermore oriented with its transcript exit channel facing a small channel through the lambda 1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein lambda 2.
濃度: 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
結晶化
*PLUS
手法: cryo electron microscopy / 詳細: cryo electron microscopy
-
電子顕微鏡撮影
EM imaging
電子線源: FIELD EMISSION GUN / 照射モード: FLOOD BEAM / モード: BRIGHT FIELD / Specimen-ID: 1
ID
加速電圧 (kV)
倍率(補正後) (X)
日付
モデル
Cs (mm)
最大 デフォーカス(公称値) (nm)
最小 デフォーカス(公称値) (nm)
倍率(公称値) (X)
温度 (K)
傾斜角・最大 (°)
傾斜角・最小 (°)
1
200
39200
1997年12月1日
FEI/PHILIPS CM200T
2
3200
1300
38000
97
0
0
2
300
47440
2002年4月1日
FEI/PHILIPS CM300FEG/T
45000
撮影
ID
Imaging-ID
電子線照射量 (e/Å2)
フィルム・検出器のモデル
1
1
20
KODAK SO-163 FILM
2
2
KODAK SO-163 FILM
画像スキャン
デジタル画像の数: 54
-
解析
EMソフトウェア
名称: PFT / カテゴリ: 3次元再構成 / 詳細: PFT2D
CTF補正
詳細: INDIVIDUAL PARTICLES, PHASE AND AMPLITUDE
対称性
点対称性: I (正20面体型対称)
3次元再構成
手法: SPHERICAL HARMONICS / 解像度: 7.6 Å / 粒子像の数: 7939 / ピクセルサイズ(公称値): 2.3 Å / ピクセルサイズ(実測値): 2.21 Å / 倍率補正: CROSS-CORRELATION 詳細: STRUCTURE OF REOVIRUS CORE (REINISCH ET AL. 2000 NATURE 404:960-967) (PDB ENTRY 1EJ6). PROTEIN ATOMS 9986, NUCLEIC ACID ATOMS 281, HETEROGEN ATOMS 86, SOLVENT ATOMS 350. 対称性のタイプ: POINT
原子モデル構築
プロトコル: RIGID BODY FIT 詳細: METHOD--SITUS (CHACON ET AL, 2002 J. MOL. BIOL. 317,375-394) WAS USED TO FIT THE X-RAY STRUCTURE OF REOVIRUS POLYMERASE (TAO ET AL. 2002 CELL 111, 733-745) (PDB CODE 1N35) INTO THE EM DENSITY ...詳細: METHOD--SITUS (CHACON ET AL, 2002 J. MOL. BIOL. 317,375-394) WAS USED TO FIT THE X-RAY STRUCTURE OF REOVIRUS POLYMERASE (TAO ET AL. 2002 CELL 111, 733-745) (PDB CODE 1N35) INTO THE EM DENSITY OF THE 7.6-A RESOLUTION MAP OF REOVIRUS VIRIONS. THE COORDINATES OF THE FITTED REOVIRUS POLYMERASE WERE ALIGNED TO THE X-RAY STRUCTURE OF REOVIRUS CORE (REINISCH ET AL. 2000 NATURE 404,960-967) (PDB ENTRY 1EJ6). ALL MATRICES FOR BUILDING AN ICOSAHEDRON CAN BE FOUND IN 1EJ6.