crystal structure reconstruction
Hi, I am new to Chimera. I have a PDB of a protein comprising of a loop region that appears to have slight alpha-helical behavior when visualized in Pymol. However, I doubt that this region is actually structured, and that this alpha-helical behavior is actually an artifact from experimental conditions. To double-check the validity of this loop, I was broadly advised that "crystal structure reconstruction" can be performed in Chimera. I suspect this is either the 'molmap' or "Fit in Map" feature? Sincerely, Lili
Hi Lili, I'm not sure what the person advising you meant -- I don't think it would make any sense to use molmap, since that would just simulate a density map from the coordinates, which are what you are unsure about in the first place. My only ideas for examining this possibly dubious helical assignment are: (a) look at B-factor values for the loop, which will give some idea of disorder, for example command: rangecolor bfactor min blue mid red max yellow (or see graphical interface "Render by Attribute" under Tools... Structure Analysis. It shows a histogram of the values.) <http://www.cgl.ucsf.edu/chimera/docs/UsersGuide/tutorials/bfactor.html> However, if the loop is constrained by the crystal structure, it may still have low B-factors, and ** there is nothing in Chimera that will predict for you the intrinsic flexibility of the loop **. I believe you would need to use some other program that simulates motions or uses some empirical criteria to predict flexibility. ... if your doubts are more toward whether the coordinates are really helical or whether they well represent the original density map, the following may be useful ... (b) re-run secondary structure assignment calculations in Chimera. Different methods produce partially different results even for exactly the same atomic coordinates because their criteria differ, so where the authors thought there was a helix, other methods might not. If you open Model Panel (from Favorites menu) there is a "compute SS" button on the right with which you can try recalculating secondary structure. <http://www.cgl.ucsf.edu/chimera/docs/UsersGuide/modelpanel.html> (c) if your structure is from the Protein Data Bank, you may be able to also get the corresponding experimental electron density map and see how well it agrees with the coordinates in the PDB file. menu: File... Fetch by ID, database: EDS (2fo-fc), enter the same 4 digits as the PDB entry. However, not all PDB entries have corresponding maps at EDS. <http://www.cgl.ucsf.edu/chimera/docs/UsersGuide/fetch.html> <http://eds.bmc.uu.se/eds/index.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 Sep 29, 2012, at 5:07 PM, rainy908@yahoo.com wrote:
Hi, I am new to Chimera. I have a PDB of a protein comprising of a loop region that appears to have slight alpha-helical behavior when visualized in Pymol. However, I doubt that this region is actually structured, and that this alpha-helical behavior is actually an artifact from experimental conditions. To double-check the validity of this loop, I was broadly advised that "crystal structure reconstruction" can be performed in Chimera. I suspect this is either the 'molmap' or "Fit in Map" feature? Sincerely, Lili
Hi Elaine, Thank you for the detailed suggestions. Following your directions, I re-ran secondary structure calculations in Chimera, and the structure of the loop region did not change. Unfortunately, there is no experimental density map corresponding to the PDB. I performed B-factor analysis on the entire protein, and interestingly, the loop region has the highest B-factor values (yellow) of out all the other regions, including other loops on the protein. I'd like to clarify that this alpha-helical region corresponds to only 2 amino acids (the entire loop is 9 residues). Given the high B-factor of the loop, if I were to run an MD simulation on this protein to witness conformational changes (~10 ns), I would suspect that the atomic positions of the loop would change anyway? In other words, the ordered behavior of the 2 residues is transient? Thanks again! Lili On 30 September 2012 10:47, Elaine Meng <meng@cgl.ucsf.edu> wrote:
Hi Lili, I'm not sure what the person advising you meant -- I don't think it would make any sense to use molmap, since that would just simulate a density map from the coordinates, which are what you are unsure about in the first place.
My only ideas for examining this possibly dubious helical assignment are:
(a) look at B-factor values for the loop, which will give some idea of disorder, for example command: rangecolor bfactor min blue mid red max yellow (or see graphical interface "Render by Attribute" under Tools... Structure Analysis. It shows a histogram of the values.) <http://www.cgl.ucsf.edu/chimera/docs/UsersGuide/tutorials/bfactor.html>
However, if the loop is constrained by the crystal structure, it may still have low B-factors, and ** there is nothing in Chimera that will predict for you the intrinsic flexibility of the loop **. I believe you would need to use some other program that simulates motions or uses some empirical criteria to predict flexibility.
... if your doubts are more toward whether the coordinates are really helical or whether they well represent the original density map, the following may be useful ...
(b) re-run secondary structure assignment calculations in Chimera. Different methods produce partially different results even for exactly the same atomic coordinates because their criteria differ, so where the authors thought there was a helix, other methods might not. If you open Model Panel (from Favorites menu) there is a "compute SS" button on the right with which you can try recalculating secondary structure. <http://www.cgl.ucsf.edu/chimera/docs/UsersGuide/modelpanel.html>
(c) if your structure is from the Protein Data Bank, you may be able to also get the corresponding experimental electron density map and see how well it agrees with the coordinates in the PDB file. menu: File... Fetch by ID, database: EDS (2fo-fc), enter the same 4 digits as the PDB entry. However, not all PDB entries have corresponding maps at EDS. <http://www.cgl.ucsf.edu/chimera/docs/UsersGuide/fetch.html> <http://eds.bmc.uu.se/eds/index.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 Sep 29, 2012, at 5:07 PM, rainy908@yahoo.com wrote:
Hi, I am new to Chimera. I have a PDB of a protein comprising of a loop region that appears to have slight alpha-helical behavior when visualized in Pymol. However, I doubt that this region is actually structured, and that this alpha-helical behavior is actually an artifact from experimental conditions. To double-check the validity of this loop, I was broadly advised that "crystal structure reconstruction" can be performed in Chimera. I suspect this is either the 'molmap' or "Fit in Map" feature? Sincerely, Lili
Hi Lili, I can't predict what an MD simulation might show for your protein -- while higher B-factors suggest higher flexibility, you would need to exercise your own scientific judgment on that, or actually perform the experiment (MD). Chimera doesn't do MD calculations, so you'd have to use some other program. Best, 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 Sep 30, 2012, at 5:07 PM, rainy908@yahoo.com wrote:
Hi Elaine,
Thank you for the detailed suggestions. Following your directions, I re-ran secondary structure calculations in Chimera, and the structure of the loop region did not change. Unfortunately, there is no experimental density map corresponding to the PDB.
I performed B-factor analysis on the entire protein, and interestingly, the loop region has the highest B-factor values (yellow) of out all the other regions, including other loops on the protein. I'd like to clarify that this alpha-helical region corresponds to only 2 amino acids (the entire loop is 9 residues).
Given the high B-factor of the loop, if I were to run an MD simulation on this protein to witness conformational changes (~10 ns), I would suspect that the atomic positions of the loop would change anyway? In other words, the ordered behavior of the 2 residues is transient?
Thanks again! Lili
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rainy908＠yahoo.com