Hi Elaine, Thank you so much for your reply. I did what you said in the letter. However the return is "Rotation angle is near zero". Then no axis came out. What I want to do is to get the elbow angle of the Fab. The elbow angle is the angle between pseudo 2-fold axes relating heavy and light chains in the variable domain and heavy and light chains in the constant domain. So I have to make two axes for variable and constant domain respectively. Then measure the angles. Looking forward to your reply. Thanks, Jian

-----Original Message----- From: Elaine Meng [mailto:meng@cgl.ucsf.edu] Sent: 2013年12月27日 13:11 To: Jian Guan Cc: chimera-users@cgl.ucsf.edu Subject: Re: [Chimera-users] Find pseudo 2-fold axes relating heavy and light chain

Hi Jian, Define Axes creates a best-fit axis to the atoms you specify. It is not meant to find axes of symmetry or pseudosymmetry.

I would recommend opening two copies of your structure, then superimposing them based on the parts that are pseudosymmetric (the parts that are pseudoequivalent), then using command "measure rotation" with option "showAxis true" (or maybe "showSlabs true") to show the axis of rotation between the two copies: http://www.rbvi.ucsf.edu/chimera/docs/UsersGuide/midas/measure.html#rotatio n

Ways to superimpose structures are discussed here: http://www.rbvi.ucsf.edu/chimera/docs/UsersGuide/superposition.html

I hope this helps, Elaine ----- Elaine C. Meng, Ph.D. UCSF Computer Graphics Lab (Chimera team) and Babbitt Lab Department of Pharmaceutical Chemistry University of California, San Francisco

On Dec 27, 2013, at 8:08 AM, Jian Guan wrote:

Dear all, I am trying to find pseudo 2-fold axes relating heavy and light chains in the constant domain and variable domain respectively in Fab. I tried Define Axes, it failed. And I made artificial double heavy chain, it failed again. I can only get axis which is horizontal passing through both light chain and heavy

chain.

It is not what I want. Can anyone help to give some clues? Thanks Jian

I improved the Chimera "angle" command so it can report the angle between any two bonds even if they do not share an endpoint. This can replace steps 3 and 4 of the procedure below for measuring the angle between axes. Instead you would select the end-points of the two axes created by "measure rotation" and then use

angle sel dihedral false

In general, the angle command with the new option "dihedral false" computes the angle between vector 12 an and vector 34 when 4 atoms are specified, for example,

angle :47@CA :52@CA :121@CA :115@CA dihedral false

computes the angle between the vector from residue 47 to 52 and vector from residue 121 to 115. Without the "dihedral false" option it reports the dihedral angle.

Tom

On Dec 27, 2013, at 11:48 AM, Elaine Meng wrote:

Hi Jian, Most important is to make sure you have superimposed the correct (pseudoequivalent) parts. My earlier answer was for calculating one pseudosymmetry axis. From what you say, and also from this descriptive page I found, you need to calculate two of those, and then measure the angle between them. http://proteinmodel.org/AS2TS/RBOW/calculation.htm

I was able to do it in Chimera and reproduce the result for the example structure 1bbd in that page. My calculation gave 53 degrees (because the axes in the calculation don't have a direction), whereas the real answer is 180 - 53 = 127 degrees as stated in that page. You can see by just looking at the structure whether it is 53 or 127.

(1) open two copies, superimpose V-H of one copy onto V-L of the other copy, use "measure rotation" to show that pseudo 2-fold axis. The tricky part is only using the V (variable) parts of the chains instead of the whole H and L chains when superimposing the structures. More detail on that below.

(2) next superimpose the C-H of one copy onto C-L of the other copy, use "measure rotation" to show that pseudo 2-fold axis.

(3) now you have two axes, but they are defined as marker sets by the "measure" command and there is no direct way to get the angle between them... so it is necessary to use Define Axes to copy them as axes that can be used in measurements. My Fab copies were models #0,1 and the axes from measure were models #2,3. I selected #2 (for example, command: select #2) and then used define axis for the selected atoms (markers are fake atoms), then selected #3 and used define axis again for the selected atoms, making sure to turn OFF the option to replace the existing define-axis.

(4) then in the table of Axes/Planes/Centroids, two axes are listed. Use the mouse to choose those two rows in the table, which automatically reports the axes' angle of intersection near the bottom of that dialog. Image of what I had then is attached below.

OK, now the detail about how to only use parts of the chains in the matches. I opened 1bba two times, so it was models #0 and #1. Each model has chains H (heavy) and L (light). I just used Ctrl-drag in the Chimera window to drag a selection box only around the variable domains. Then in MatchMaker I chose "Chain pairing: Specific chain(s) in reference structure with specific chain(s) in match structure" with reference chain #0 chain L and chain to match #1 chain H, and also checked the box "Further restrict matching to current selection." Thus I could superimpose #0 and #1 using only V-H and V-L. After using "measure rotation" to generate the first axis, I chose from the main Chimera menu "Select... Invert (selected models)" so now only the constant parts are selected, and used Matchmaker again without changing any of the settings in that dialog. Then used "measure rotation" ... etc. The main thing is to draw the first selection box carefully. It doesn't matter if you draw it around variable or constant, because the other one will be calculated after you invert selection.

I don't have any ideas about what to do if the angle is near zero, but if you really superimposed the structures correctly, my guess is it should not be near zero.

Well, now I've learned a lot (too much?) about Fab structures! Elaine

<grab.tiff>

On Dec 27, 2013, at 10:40 AM, Jian Guan wrote:

...

Hi Elaine, Thank you so much for your reply. I did what you said in the letter. However the return is "Rotation angle is near zero". Then no axis came out. What I want to do is to get the elbow angle of the Fab. The elbow angle is the angle between pseudo 2-fold axes relating heavy and light chains in the variable domain and heavy and light chains in the constant domain. So I have to make two axes for variable and constant domain respectively. Then measure the angles. Looking forward to your reply. Thanks, Jian

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