CS/CAM/BME 395T - #54303

Multi-scale Bio-Modeling and Visualization

 

Exercise #2: Segmentation and visualization

 

Each student shall be assigned three maps to work on.

 

The steps to segment each map (I am using the example emd_1011.map):

 

1. Go to the following link and download the maps which you are assigned.

http://www.ebi.ac.uk/msd-srv/emsearch/AccEMServ?criterion=Author&value1=&andor=and&value2=&Submit=Submit+Query

 

 2. Unzip the map. Run VolRover (you need to download VolRover from CVC webpage). Read the map. Adjust the little cube in the right window to the maximal size.  Save the Subvolume as `rawiv` type.

 

3. In VolRover, you also need to decide visually the h and k numbers (h_num and k_num) of the virus structure. If you have difficulty in finding these numbers, please check the reference of that map in the above link, where you may find this information. You need to decide if there is a 5-fold subunit (if yes, the parameter 5-fold = 1; otherwise, 0). You also need to decide whether the major subunits to be segmented are located at the 3-fold grids or 6-fold grids (see Figure 1). If the subunits are at the 3-fold grids (red dots), 6-fold = 0 and 3-fold is equal to the folding number of the subunits. If the subunits are at the 6-fold grids (blue dots), 3-fold = 0 and 6-fold is equal to the folding number of the subunits. Again, reading the reference would be helpful for you to decide these parameters.

 

 

 

Figure1 The blue dots are 6-fold grids and the red dots are the 3-fold grids.

 

 

 

4. Copy the rawiv map (named emd_1011.rawiv, for instance) to your working directory on maverick.tacc.utexas.edu.

 

5. Compile the makeslice_z.c by `cc makeslice_z.c -o makeslice_z`.

 

6. Run makeslice_z as follows:

            makeslice_z emd_1011.rawiv tt.ppm slice_num

slice_num is usually half the dimension in z-axis. If you do not know the dimension, try zero and makeslice_z will tell you the dimension of the map. The following figure shows the central slice of emd_1011.rawiv.

 

Figure 2

 

7. Type `xv tt.ppm` on maverick and you will see the above figure. Use the middle button of the mouse to select the threshold tlow for the capsid layer. It is very important to choose a good threshold for correct segmentation of the capsid layer.

8. Compile the segmentation source code. Run the segmentation using the parameters determined above:

 

CCVseg3D emd_1011.rawiv tlow h_num k_num 3-fold 5-fold 6-fold

 

The segmentation tool will return the following files:

  • emd_1011_matrix.txt: transformation matrices
  • emd_1011_5f_subunit.rawiv: segmented 5-fold subunit
  • emd_1011_6f_subavg.rawiv: average of segmented 6-fold subunits
  • emd_1011_seg.rawv: color map of segmentation
  • emd_1011_index.rawiv: indexing map of segmentation
  • symmetry_axis.raw: symmetry axes
  • comatrix.txt: correlation scores
  • six_fold.txt: returned 6-fold symmetry axes
  • five_fold.txt: returned 5-fold symmetry axes

 

 

9. Display/Edit the results:

  • Symmetry axes: Run VolRover. Open emd_1011.rawiv. From the menu `Geometry`, choose `Load Geometry`. Select the file `symmetry_axis.raw` that the segmentation returned just now. You will see the following picture. Save it.

 

Figure 3.

 

Important! When making your pictures, please set the background as white (in `File`->`Option`->`Background color`). Disable the wire cube (in `View`->`Show Wire Cube`). Same for the following pictures.

 

 

  • Segmentation color map: Run VolRover and open `emd_1011_seg.rawv`. From the menu `File`, go to `Options..` and select the `RGBA Combined`. You will see the following picture (the left one). In VolRover, you can see a few adjustable bars below the left and right windows. Adjust the `Explorer Near Clip Plane` just below the right window. You will see the segmented subunits from the inside (see the picture below on the right-hand side). Save both pictures.

 

 

 

Figure 4

 

 

  • Segmented 5-fold subunit: Run VolRover and open `emd_1011_5f_subunit.rawiv`. In the color window (on the bottom of VolRover), Click the right button of mouse, Go to `Add`->`Isocontour Node`. Adjust the node to change the isovalue. Once a good isovalue is selected, save the picture (see example in Figure 5).

 

         

 

Figure 5

 

 

  • Average of segmented 6-fold subunits: Run VolRover and open `emd_1011_6f_subavg.rawiv`. Choose a good isovalue and save the picture (see example in Figure 6).

 

       

 

Figure 6

 

  • Similarity Table:

From `comatrix.txt`, we can find the similarity scores between subunits:

1.000000 0.000000 0.000000 0.000000

0.887794 -0.800000 3.141592 0.368794

0.907930 -0.400000 -0.017453 0.190471

0.889730 -1.100000 1.064651 0.297578

 

-1.000000 0.800000 -3.141592 0.368794

1.000000 0.000000 0.000000 0.000000

0.899031 0.400000 -3.176499 0.526629

0.875711 -0.400000 -2.076942 0.201592

 

-1.000000 0.400000 0.017453 0.190471

-1.000000 -0.400000 3.176499 0.526629

1.000000 0.000000 0.000000 0.000000

0.882088 -0.600000 1.134464 0.391825

 

-1.000000 1.100000 -1.064651 0.297578

-1.000000 0.400000 2.076942 0.201592

-1.000000 0.600000 -1.134464 0.391825

1.000000 0.000000 0.000000 0.000000

 

 

1

2

3

4

1

 

0. 887794

0.907930

0.889730

2

0. 887794

 

0.899031

0.875711

3

0.907930

0.899031

 

0.882088

4

0.889730

0.875711

0.882088

 

 

From `six_fold.txt` (`three_fold.txt`, if the subunits have 3-fold symmetry), we can find the symmetry scores for each subunit (meaning the similarity score between a subunit and its rotated copies):

128.175659 150.750244 228.367996 126.221344 139.842041 189.933731 0.940744

...... (59 more)

135.938095 125.366867 233.145020 138.974777 128.185486 193.360168 0.909652

...... (59 more)

152.750320 152.618988 227.991394 144.223923 138.450409 191.569519 0.936145

...... (59 more)

161.008606 126.777542 233.774170 156.319061 127.336990 194.053955 0.907511

...... (59 more)

 

 

1

2

3

4

1

0.940744

 

     

 

2

 

0.909652

 

 

3

 

 

0.936145

 

4

 

 

 

0.907511

 

Merge the above two tables, we can have the similarity table:

 

 

1

2

3

4

1

0.940744

0. 887794

0.907930

0.889730

2

0. 887794

0.909652

0.899031

0.875711

3

0.907930

0.899031

0.936145

0.882088

4

0.889730

0.875711

0.882088

0.907511

 

Table 1

10. Deposit your results:

The following files shall be submitted:

  • Pictures (Figures 3-6) and Table 1.
  • Segmentation results:
    • emd_1011_matrix.txt: transformation matrices
    • emd_1011_5f_subunit.rawiv: segmented 5-fold subunit
    • emd_1011_6f_subavg.rawiv: average of segmented 6-fold subunits
    • emd_1011_seg.rawv: color map of segmentation
    • emd_1011_index.rawiv: indexing map of segmentation
    • symmetry_axis.raw: symmetry axes
    • comatrix.txt: correlation scores
    • six_fold.txt: returned 6-fold symmetry axes
    • five_fold.txt: returned 5-fold symmetry axes

Important: remember to change the names of the last four files to avoid overwriting. (I'll change the output names of these files in later version).

  • PDF of the reference and give the reference and a pointer to the PDF.

They should be zipped and uploaded to any of your websites and you email Dr. Bajaj (bajaj@cs.utexas.edu) the URL. If you do not have access to a webspace, please contact Jason (jgsun@ices.utexas.edu) for an alternative method to upload your results.

 

 

 

 

Summary: EBI cryo-EM icosahedral maps

 

Red: structures which have been segmented.

Blue: structures which are unavailable at this time

Others: for the student to work on (26 maps)

 

1011

icosahedral

Bacteriophage PRD1

16

2002-10-09

 

C. San Martin, J. T. Huiskonen, J. K. Bamford, S. J. Butcher, S. D. Fuller, D. H. Bamford, R. M. Burnett

1012

icosahedral

Bacteriophage PRD1 sus607 mutant

13.4

2002-10-10

 

C. San Martin, J. T. Huiskonen, J. K. Bamford, S. J. Butcher, S. D. Fuller, D. H. Bamford, R. M. Burnett

1013

icosahedral

Bacteriophage PRD1 sus1 mutant

14

2002-10-10

 

C. San Martin, R. M. Burnett, de Haas, R. Heinkel, T. W. Rutten, S. D. Fuller, S. J. Butcher, D. H. Bamford

1014

icosahedral

Bacteriophage PRD1 P3 shell

12

2002-10-10

 

C. San Martin , R. M. Burnett, F. de Haas, R. Heinkel, T. Rutten, S. D. Fuller, S. J. Butcher, D. H. Bamford

1015

icosahedral

Semliki Forest Virus

9.0

2002-10-11

 

E. J. Mancini, M. Clarke, B. Gowen, T. Rutten, S. D. Fuller,

1016

icosahedral

adenovirus type 2

25

2002-10-15

 

P.L. Stewart, S.D. Fuller, R. M. Burnett

1018

icosahedral

Semliki forest mSQL

9.0

2003-01-06

2003-02-12

I.Ferlenghi,B. Gowen B,F. de Haas F, EJ Mancini, H. Garoff, M.Sjoberg, S.D. Fuller

1049

icosahedral

Human Rhinovirus 2 in complex with its cellular receptor, VLDL-R

16

2003-06-03

2003-05-19

E. Neumann, R. Moser, L.Snyers, D. Blaas and E.A. Hewat

1057

icosahedral

Echovirus type 12 bound to decay accelerating factor domains 3 and 4

16

2003-11-25

2003-10-08

D. Bhella, I.G. Goodfellow, P. Roversi, D. Pettigrew,Y.Chaudhry, D.J. Evans, S.M. Lea

1058

icosahedral

Echovirus type 12

18

2003-11-25

2003-10-08

D. Bhella, I.G. Goodfellow, P. Roversi, D. Pettigrew, Y. Chaudhry, D.J. Evans, S.M. Lea

1060

icosahedral

Rice Dwarf Virus

6.8

2005-04-08

2003-10-21

Zhou ZH, Baker ML, Jiang W, Dougherty M, Jakana J, Dong G, Lu G, Chiu W.

 1082

icosahedral

PM2 virion

8.4

2005-05-25

2004-05-25

J.T. Huiskonen, H.M. Kivela, D.H. Bamford, S.J. Butcher

1083

icosahedral

PM2 virion

12.3

2005-05-25

2004-05-25

J.T. Huiskonen, H.M. Kivela, D.H. Bamford, S.J. Butcher

1084

icosahedral

Proteinase K-treated PM2 virus

13

2005-05-25

2004-05-25

J.T. Huiskonen, H.M. Kivela, D.H. Bamford, S.J. Butcher

1085

icosahedral

Bromelain-treated PM2 virus

13.4

2005-05-25

2004-05-25

J.T. Huiskonen, H.M. Kivela, D.H. Bamford, S.J. Butcher

1101

icosahedral

P22 Mature Phage

9.5

2004-12-13

2004-12-13

W. Jiang, Z. Li, Z. Zhang, M.L. Baker, P.E. Jr Prevelige, W. Chiu

1111

icosahedral

Human adenovirus type 5 pIX deletion mutant

22

2006-06-07

2005-02-25

Celine MS Fabry, Manuel Rosa-Calatrava, James Conway, Chloe Zubieta, Stephen Cusack, Rob WH Ruigrok, Guy Schoehn

1112

icosahedral

Human adenovirus type 5 pIX deletion mutant

15.5

2005-06-07

2005-02-25

Celine MS Fabry, Manuel Rosa-Calatrava, James Conway, Chloe Zubieta, Stephen Cusack, Rob WH Ruigrok, Guy Schoehn

1113

icosahedral

Human adenovirus type 5

10.3

2005-06-07

2005-02-25

Celine MS Fabry, Manuel Rosa-Calatrava, James Conway, Chloe Zubieta, Stephen Cusack, Rob WH Ruigrok, Guy Schoehn

1114

icosahedral

Human Coxsackievirus A21 complex with ICAM-1KilifiFc

8.0

2006-03-29

2005-03-29

C.Xiao, C.M.Bator-Kelly, E.Rieder, P.R.Chipman, A.Craig, R.J.Kuhn, E.Wimmer, M.G.Rossmann

1115

icosahedral

Infectious Bursal Disease Virus capsid

12

2006-03-30

2005-03-30

I. Saugar, D. Luque, A. Ona, J.F. Rodriguez, J. L. Carrascosa, B. L. Trus, J R. Caston

1116

icosahedral

fibered isometric phi29 particle

8.7

2005-04-26

2005-03-30

M.C. Morais, K.H. Choi, J.S.Koti, P.R. Chipman, D.L. Anderson, M.G. Rossmann

1118

icosahedral

chimeric Infectious Bursal Disease Virus capsid

15

2006-03-31

2005-03-31

I. Saugar, D. Luque, A. Ona, J.F. Rodriguez, J. L. Carrascosa, B. L. Trus, J R. Caston

1120

icosahedral

phi29 fiberless isometric particle

7.9

2005-04-26

2005-04-05

M.C. Morais, K.H. Choi, J.S. Koti, P.R. Chipman, Anderson, D.L., Rossmann, M.G.

1121

icosahedral

Sindbis TE12 E2-N318Q

9.0

2006-04-08

2005-04-08

S. Mukhopadhyay, W. Zhang, S. Gabler, P. R. Chipman, E. G. Strauss, J. H. Strauss, T. S. Baker, R. J. Kuhn, M. G. Rossman

1123

icosahedral

Bam35c virion

7.4

2006-04-27

2005-04-27

P. A. Laurinmaki, J. T. Huiskonen, D. H. Bamford, S. J. Butcher

1124

icosahedral

Bam35c empty virion

18

2006-04-27

2005-04-27

P. A. Laurinmaki, J. T. Huiskonen, D. H. Bamford, S. J. Butcher

1133

icosahedral

Poliovirus 135S particle

9.6

2005-06-07

2005-06-02

Bubeck D, Filman DJ, Cheng N, Steven AC, Hogle JM, Belnap DM

1136

icosahedral

Poliovirus 135S particle

8.7

2005-07-21

2005-07-15

Bubeck D, Filman DJ, Cheng N, Steven AC, Hogle JM, Belnap DM.

1137

icosahedral

Poliovirus 135S particle

10.3

2005-07-21

2005-07-15

Bubeck D, Filman DJ, Cheng N, Steven AC, Hogle JM, Belnap DM.

1144

icosahedral

Poliovirus 135S particle

11

2005-09-02

2005-08-04

D. Bubeck, D.J. Filman, N. Cheng, A.C. Steven, J.M. Hogle, D.M. Belnap

1145

icosahedral

Poliovirus 135S particle

11

2005-09-02

2005-08-04

D. Bubeck, D.J. Filman, N. Cheng, A.C. Steven, J.M. Hogle, D.M. Belnap

1150

icosahedral

Kelp fly virus

18.0

2005-09-02

2005-08-10

C.J.Hartley, D.R.Greenwood, R.J.C.Gilbert,

1153

icosahedral

Kelp fly virus

15.0

2005-09-02

2005-08-17

C.J.Hartley, D.R.Greenwood, R.J.C.Gilbert,

1154

icosahedral

Kelp fly virus

15.0

2005-09-02

2005-08-17

C.J.Hartley, D.R.Greenwood, R.J.C.Gilbert,

 

 

Here is the assignment sheet.

 

 

Questions? Email to Zeyun Yu: zeyun.yu@gmail.com