近年來Cyro-EM 與影像重建，已經成為檢驗病毒與研究病毒的結構的主要工具，這種鑑定結構的方式具有快速，且能保持樣品原有型態的特點。使用軟體方式模擬三種病毒DGNNV、PaV 以及TBSV 的影像具有相當極端的偏好方位，再使用SPIDER 以及PurdueEM 以投影比對的方式來重建模型。SPIDER 偏好三重與五重對稱軸端視角，
PurdueEM 偏好二重對稱軸端視角。SPIDER 可以重建出加了與
PurdueEM 相比較多量雜訊的影像。RNA-cage 的結構跟病毒外殼相關。偏好方位、雜訊以及RNA 這三種視角取向均會影響病毒顆粒三維重建。

Cyro-EM and three-dimensional reconstruction have become important research tools for virus structure. These techniques have benefit of fast and keep samples in native folding. Simulated prefer orientation images of DGNNV, PaV and TBSV were reconstructed by SPIDER or PurdueEM, with projection matching with. SPIDER prefers 3-fold and 5-fold view fields, and PurdueEM prefers 2-fold view fields. SPIDER could reconstruction images which have more noise than PurdueEM can successfully reconstruct. Reconstruction of RNA-cages has some
relationship with the symmetry of capsid protein. Prefer orientation, noise and RNA-cages are the factors that can effect reconstruction.

摘要......................................................................................................................................................... I
ABSTRACT ........................................................................................................................................ III
目錄...................................................................................................................................................... IV
圖目錄.................................................................................................................................................. VI
壹、前言.................................................................................................................................................1
一、 電子顯微鏡...........................................................................................................................1
二、 冷凍電子顯微鏡...................................................................................................................3
三、 單一顆粒影像重建原理.......................................................................................................4
1. 共通線..................................................................................................................................5
2. Random-conical.....................................................................................................................6
3. 投影比對...............................................................................................................................6
四、 立體重建原理.......................................................................................................................6
1. 權衡式反投影.......................................................................................................................7
2. 傅立葉重建...........................................................................................................................7
五、 影像重建軟體與重建流程...................................................................................................7
A. EMAN..................................................................................................................................7
B. PurdueEM...........................................................................................................................12
C. SPIDER..............................................................................................................................16
六、 近期發展況狀.....................................................................................................................17
七、 偏好方位.............................................................................................................................21
貳、材料與方法...................................................................................................................................23
一、 測試軟體版本.....................................................................................................................23
二、 製作測試影像.....................................................................................................................23
1. 測試模型製作.....................................................................................................................23
2. 設定投影角度.....................................................................................................................23
3. 投影測試影像.....................................................................................................................23
5. 建立初始模型.....................................................................................................................24
三、 檔案格式轉換.....................................................................................................................24
1. 平面影像.............................................................................................................................25
2. 平面影像：其他格式轉換至PurdueEM...........................................................................25
3. 平面影像：PurdueEM (PIF)至其他軟體...........................................................................26
4. 立體模型.............................................................................................................................26
四、 PurdueEM 影像重建...........................................................................................................26
五、 SPIDER 影像重建..............................................................................................................28
六、 Fouriel shell correlation.......................................................................................................33
七、 數據統計.............................................................................................................................33
八、 偏好方位測試.....................................................................................................................34
九、 雜訊....................................................................................................................................34
十、 RNA-cage...........................................................................................................................34
參、結果...............................................................................................................................................35
一、 正二十面體角度定義.........................................................................................................35
二、 DGNNV 偏好方位測試......................................................................................................35
三、 PaV 偏好方位測試.............................................................................................................38
四、 TBSV 偏好方位測試..........................................................................................................38
五、 雜訊....................................................................................................................................40
六、 RNA-cage...........................................................................................................................41
肆、討論...............................................................................................................................................44
一、 偏好方位測試.....................................................................................................................44
二、 雜訊....................................................................................................................................49
三、 RNA-cage...........................................................................................................................50
伍、參考文獻.......................................................................................................................................52
陸、圖表...............................................................................................................................................58
Fig. 1 Definition of view orientation angles of asymmetric unit in an
icosahedral virion.................................................................................58
Fig. 2 3D models of DGNNV, PaV and TBSV............................................59
Fig. 3 Reference images of DGNNV...........................................................61
Fig. 4 References of PaV .............................................................................63
Fig. 5 References of TBSV..........................................................................65
Fig. 6 View field along 2-5-lateral reconstruction by SPIDER of DGNNV.67
Fig. 7 View field along 2-5-lateral within 14o -25o reconstruction by
SPIDER of DGNNV.............................................................................69
Fig. 8 View field along 2-5-lateral reconstruction by PurdueEM of DGNNV.
..............................................................................................................71
Fig. 9 View field along 2-5-lateral with in 10o -18o reconstruction by
PurdueEM of DGNNV. ........................................................................73
Fig. 10 View field along 2-3-lateral reconstruction by PurdueEM of
DGNNV................................................................................................75
Fig. 11 View field along 3-5-lateral reconstruction by SPIDER of DGNNV.
..............................................................................................................77
Fig. 12 View field along 3-5-lateral reconstruction by PurdueEM of
DGNNV................................................................................................79
Fig. 13 View field along 2-5-lateral reconstruction by SPIDER of PaV......81
Fig. 14 View field along 2-5-lateral reconstruction by PurdueEM of PaV. .83
Fig. 15 View field along 2-5-lateral reconstruction by SPIDER of TBSV. .85
Fig. 16 View field along 2-5-lateral reconstruction by PurdueEM of TBSV.
..............................................................................................................87
Fig. 17 View field along 2-3-lateral reconstruction by SPIDER of TBSV. .89
Fig. 18 View field along 2-3-lateral reconstruction by PurdueEM of TBSV.
..............................................................................................................91
Fig. 19 View field along 3-5-lateral reconstruction by SPIDER of TBSV. .93
Fig. 20 View field along 3-5-lateral reconstruction by PurdueEM of TBSV.
..............................................................................................................95
Fig. 21 FSC curve of DGNNV maps reconstructed SPIDER and PurdueEM.
..............................................................................................................96
Fig. 22 FSC curve of PaV maps reconstructed by SPIDER and PurdueEM.
..............................................................................................................97
Fig. 23 FSC curve of TBSV maps reconstructed by SPIDER and
PurdueEM. ...........................................................................................98
Fig. 24 Noise effects on 3D-reconstruction of DGNNV by SPIDER..........99
Fig. 25 Noise effects on 3D-reconstruction of DGNNV by PurdueEM. ...100
Fig. 26 Noise effects on 3D-reconstruction of PaV by SPIDER. ..............101
Fig. 27 Noise effects on 3D-reconstruction of PaV by PurdueEM............102
Fig. 28 Noise effects on 3D-reconstruction of TBSV by SPIDER............103
Fig. 29 Noise effects on 3D-reconstruction of TBSV by PurdueEM. .......104
Fig. 30 Reconstruction of DGNNV RNA-cage 2-fold to 2-fold................105
Fig. 31 Reconstruction of DGNNV RNA-cage 2-fold-90o to 2-fold.........106
Fig. 32 Reconstruction of DGNNV RNA-cage with 5-fold to 2-fold. ......107
Fig. 33 Reconstruction of DGNNV RNA-cage with 3-fold to 2-fold. ......108
Fig. 34 Angular variance distribution of NNV, PaV and TBSV along
2-5-lateral...........................................................................................109
Fig. 35 Angular variance distribution of NNV, PaV and TBSV along
3-2-lateral...........................................................................................110
Fig. 36 Angular variance distribution of NNV, PaV and TBSV along
3-2-lateral........................................................................................... 111

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