I am using MR to solve the structure of a protein that is a 4-site directed mutant of the wild type(known structure, 260 amino acids). It crystallizes in a different space group (P21212121) from the native. It’s unit cell dimensions are 50.897, 94.758, 151.205 with an estimated 2 molecules in my asymmetric unit. I have used BEAST to find my rotation and translation results, but I am having some difficulty in interpreting some of the output. I appear to get clear solutions, but the model exhibits considerable steric clashes between the molecules and their symmetry related mates. I am using CCP4i, version 4.2.1 on a Compaq Alpha, Tru64.
My rotation search always gives me only 2 top rotation angles (with around 300 LLGs).
Partial output ----
Sorted list of best trials
Mol Euler angles Fractional translation LLG
BEST mol1 116.85 73.67 178.69 282.679
BEST mol1 49.66 41.01 38.95 257.279
BEST mol1 52.30 45.09 36.00 240.956
BEST mol1 114.08 77.75 181.32 179.224
BEST mol1 114.34 69.59 178.61 171.144
CLUST mol1 116.85 73.67 178.69 282.679 : 46 members
CLUST mol1 49.66 41.01 38.95 257.279 : 38 members
CLUST mol1 70.17 81.84 359.55 56.863 : 4 members
CLUST mol1 68.66 90.00 359.72 54.421 : 3 members
After a fine search about these solutions (which increases the LLGs by about 20) I do a translation search with my 2 top rotation options. I get a further increase in my LLGs.
Sorted list of best trials
Mol Euler angles Fractional translation LLG
BEST mol1 52.89 42.72 37.04 0.1346 0.2227 0.4887 351.708
BEST mol1 52.89 42.72 37.04 0.1444 0.1405 0.4887 338.858
BEST mol1 52.89 42.72 37.04 0.1346 0.2776 0.4887 337.021
BEST mol1 52.89 42.72 37.04 0.1346 0.3507 0.4887 332.362
BEST mol1 52.89 42.72 37.04 0.1444 0.3050 0.4887 325.157
BEST mol1 115.67 73.43 179.89 0.1226 0.0840 0.3668 325.893
BEST mol1 115.67 73.43 179.89 0.1226 0.0840 0.0639 323.951
BEST mol1 115.67 73.43 179.89 0.1226 0.0840 0.3252 322.643
BEST mol1 115.67 73.43 179.89 0.1226 0.0840 0.1649 316.940
BEST mol1 115.67 73.43 179.89 0.1281 0.0823 0.3639 312.382
I fix one of my rotation solutions (with its translation), rotate to my second rotation, and do a full translation search of the entire unit cell.
Sorted list of best trials
Mol Euler angles Fractional translation LLG
BEST mol1 52.89 42.72 37.04 0.8979 0.1774 0.6859 680.750
BEST mol1 52.89 42.72 37.04 0.3871 0.1774 0.2971 580.436
BEST mol1 52.89 42.72 37.04 0.8782 0.1713 0.6751 157.817
BEST mol1 52.89 42.72 37.04 0.8782 0.1591 0.6859 155.117
BEST mol1 52.89 42.72 37.04 0.3871 0.1774 0.6859 -97.172
BEST mol1 52.89 42.72 37.04 0.6425 0.4881 0.9883 -149.880
And when I did a fine search around the top rot/trans in the above list, I got an LLG of 1075…
Sorted list of best trials
Mol Euler angles Fractional translation LLG
BEST mol1 52.89 42.72 37.04 0.8891 0.1719 0.6842 1075.959
BEST mol1 52.89 42.72 37.04 0.8871 0.1725 0.6831 1059.187
BEST mol1 52.89 42.72 37.04 0.8871 0.1737 0.6842 1052.842
BEST mol1 52.89 42.72 37.04 0.8910 0.1725 0.6831 1052.695
BEST mol1 52.89 42.72 37.04 0.8910 0.1737 0.6842 1048.809
I used PDBSET to rot/trans my search model to these new orientations and I merged them using MOLEMAN. But when I look at my new merged pdb file in "O" I have significant steric clashes. The solution does not refine well using rigid body or simulated annealing refinement protocols.
Right now I am lost as to how to backtrack my steps and what takes priority over the other. Do I go back to my initial translation search of my top 2 rotations and pick the 2nd heights rot/trans value, or do I try to fix the second rotation angle, or.... If anyone would have any insight on my very frustrating problem I would truly appreciate it.
Thanks,
Jean Dehdashti
University of Missouri-Kansas City
dehdashtis@umkc.edu
P.S. I have also tried MOLREP, the ccp4 version of AMoRe and the stand-alone version of AMoRe, and get similar results.