ジャーナル: Proc Natl Acad Sci U S A / 年: 2014 タイトル: Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions. 著者: Fei Guo / Zheng Liu / Ping-An Fang / Qinfen Zhang / Elena T Wright / Weimin Wu / Ci Zhang / Frank Vago / Yue Ren / Joanita Jakana / Wah Chiu / Philip Serwer / Wen Jiang / 要旨: Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For ...Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.
A: Major capsid protein 10A B: Major capsid protein 10A C: Major capsid protein 10A D: Major capsid protein 10A E: Major capsid protein 10A F: Major capsid protein 10A G: Major capsid protein 10A
A: Major capsid protein 10A B: Major capsid protein 10A C: Major capsid protein 10A D: Major capsid protein 10A E: Major capsid protein 10A F: Major capsid protein 10A G: Major capsid protein 10A
A: Major capsid protein 10A B: Major capsid protein 10A C: Major capsid protein 10A D: Major capsid protein 10A E: Major capsid protein 10A F: Major capsid protein 10A G: Major capsid protein 10A
x 5
icosahedral pentamer
1.28 MDa, 35 ポリマー
分子量 (理論値)
分子数
合計 (水以外)
1,280,637
35
ポリマ-
1,280,637
35
非ポリマー
0
0
水
0
タイプ
名称
対称操作
数
identity operation
1_555
x,y,z
1
point symmetry operation
4
4
A: Major capsid protein 10A B: Major capsid protein 10A C: Major capsid protein 10A D: Major capsid protein 10A E: Major capsid protein 10A F: Major capsid protein 10A G: Major capsid protein 10A
名称: Bacteriophage T7 MLD capsid II / タイプ: VIRUS / 詳細: 415 copies of gp10A form T=7 icosaheral shell
分子量
値: 15.1 MDa / 実験値: NO
ウイルスについての詳細
中空か: YES / エンベロープを持つか: NO / ホストのカテゴリ: BACTERIA(EUBACTERIA) / 単離: SPECIES / タイプ: VIRION
天然宿主
生物種: Escherichia coli
緩衝液
名称: T/M buffer / pH: 7.4 / 詳細: 200 mM NaCl, 10 mM Tris-HCl, 1 mM MgCl2
試料
包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
試料支持
詳細: 400 mesh copper grid with one lacy carbon layer and one layer of ultra-thin continuous carbon film on top. The grid is then coated with poly-lysine.
急速凍結
装置: FEI VITROBOT MARK I / 凍結剤: ETHANE / Temp: 120 K / 湿度: 90 % 詳細: Blot for 2 seconds twice with 2 mm offset before plunging into liquid ethane (FEI VITROBOT MARK I). 手法: Blot for 2 seconds twice with 2 mm offset before plunging.
モード: BRIGHT FIELD / 倍率(公称値): 59000 X / 倍率(補正後): 57727 X / 最大 デフォーカス(公称値): 3300 nm / 最小 デフォーカス(公称値): 500 nm / Cs: 2.7 mm / カメラ長: 0 mm
試料ホルダ
試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 資料ホルダタイプ: Liquid nitrogen-cooled / 温度: 95 K / 最高温度: 100 K / 最低温度: 80 K
撮影
電子線照射量: 25 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM
画像スキャン
デジタル画像の数: 1368
放射
プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray
放射波長
相対比: 1
-
解析
EMソフトウェア
ID
名称
バージョン
カテゴリ
1
EMAN
1
3次元再構成
2
EMAN
2
3次元再構成
3
jspr
3次元再構成
CTF補正
詳細: Each particle
対称性
点対称性: I (正20面体型対称)
3次元再構成
手法: Projection matching / 解像度: 3.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 43417 / ピクセルサイズ(公称値): 1.1 Å / ピクセルサイズ(実測値): 1.1 Å 詳細: Particles were selected from scanned micrograph images, first automatically by the ethan method and then by manual screening with the boxer program in EMAN. The TEM instrument contrast ...詳細: Particles were selected from scanned micrograph images, first automatically by the ethan method and then by manual screening with the boxer program in EMAN. The TEM instrument contrast transfer function parameters were determined automatically using fitctf2.py and were then visually validated using the EMAN ctfit program. The datasets were then divided into two subsets (even and odd) and processed completely independently, including both initial models and refinements. For 3D reconstructions, the whole datasets were divided into even-odd halves and the initial de novo models and subsequent iterative refinements were all independently performed for each half dataset. The images were first binned 4x to obtain initial models and particle parameters assuming icosahedral symmetry. De novo initial models were built using the random model approach. Random subsets of particles were assigned random initial orientations and iteratively refined until convergence. Consistent icosahedral capsid structures (other than occasional differences in handedness) were obtained by repeating the random model process. Particles with inconsistent/unstable view parameters in the initial refinements were excluded in further image processing. The orientation and center parameters were then transferred to the un-binned images for high-resolution refinements which included Simplex method-based orientation/center optimization and grid search-based refinement of defocus, astigmatism, and magnification of the images. All image refinement and reconstructions were performed with in-house developed programs jspr.py (for overall work-flow), jalign (for 2D alignment) and j3dr (for 3D reconstruction), which use EMAN and EMAN2 library functions. 対称性のタイプ: POINT