Dear Paul Holland,
I would spend some time in improving the search model, first. If there
are more than one possible search molecules in the PDB, I usually do a
structural alignment (ssm superposition) to get an idea about the
flexibility of the search molecules. Then I remove all parts that I
suspect to be flexible (usually N- and C-termini, long loops), until I
get a very compact search model. You may try a model with no side-chain
truncation, since in the hydrophobic core, there might be a similar
isosteric packing of atoms. In addition, I would truncate the side
chains either to the longest common chain (with ctruncate) if there is a
good sequence alignment, or to poly-Ser or even poly-Ala. I would try
these "core"-models as search models, with the idea that missing atoms
lower the signal, but wrong atoms raise the noise and may produce wrong
clashes. In case of several search models, where each individual model
fails, you may try to run Phaser using all of them simultaneously as a
search ensemble.
This model-pruning together with a search-ensemble of several PDB
entries worked recently in a project, where otherwise all molecular
replacement trials failed.
Good luck,
Dirk.
Am 13.09.10 16:52, schrieb Paul Holland:
Hello fellow crystallographers,
I am trying molecular replacement for a protein crystal dataset that has very
high sequence similarity to the search model with several predicted flexible
loop regions; however, all attempts at finding a solution have not produce very
ideal starting solutions using Phaser and Molrep (CC = 0.3 and Z-score = 5). I
am very confident that the unit cell parameters are C2 84.027 120.565 108.272
90.00 104.71 90.00, and there appears to be no evidence of twinning. The
Matthews calculation predicts from anywhere from 2-4 monomers in the ASU, and
calculation of the SRF in Molrep does not identify any peaks in higher order
symmetry except for the expected crystallographic two-fold for C2. Below is
the table from the calculated SRF in molrep. Any advice would be greatly
appreciated.
# theta phi chi alpha beta gamma Rf
Rf/sigma
Sol_RF 1 0.00 0.00 0.00 0.00 0.00 0.00 870.5
21.59
Sol_RF 2 58.61 -10.17 180.00 169.83 -117.23 10.17 162.5 4.03
Sol_RF 3 66.02 -0.00 180.00 180.00 -132.03 0.00 161.1 4.00
Sol_RF 4 58.42 -9.54 180.00 170.46 -116.85 9.54 159.8 3.96
Sol_RF 5 149.84 0.00 180.00 -180.00 60.32 0.00 156.0 3.87
Sol_RF 6 58.96 -5.52 180.00 174.48 -117.91 5.52 151.5 3.76
Sol_RF 7 65.59 20.95 180.00 20.95 131.18 159.05 143.9 3.57
Sol_RF 8 90.00 -98.96 180.00 0.00 180.00 17.92 142.9 3.55
Sol_RF 9 56.53 15.78 180.00 15.78 113.07 164.22 142.0 3.52
Sol_RF 10 71.10 -19.94 180.00 160.06 -142.20 19.94 141.6 3.51
Sol_RF 11 71.28 29.78 180.00 29.78 142.55 150.22 140.4 3.48
Sol_RF 12 65.22 -15.88 180.00 164.12 -130.44 15.88 139.2 3.45
Sol_RF 13 68.84 -0.00 180.00 180.00 -137.67 0.00 138.0 3.42
Sol_RF 14 32.51 -180.00 180.00 -180.00 65.02 -0.00 137.9 3.42
Sol_RF 15 75.02 -28.84 180.00 151.16 -150.04 28.84 134.7 3.34
Sol_RF 16 71.69 -20.99 180.00 159.01 -143.37 20.99 133.0 3.30
Sol_RF 17 92.13 101.46 179.93 102.35 -175.74 79.42 130.9 3.25
Sol_RF 18 107.89 144.73 179.79 145.06 -144.22 35.61 128.8 3.19
Sol_RF 19 87.45 -78.19 180.00 101.81 -174.90 78.19 128.1 3.18
Sol_RF 20 38.57 0.69 30.36 102.66 -18.79 -78.71 122.4 3.04
Sol_RF 21 26.77 174.59 176.58 172.68 53.52 3.49 120.5 2.99
Sol_RF 22 116.66 178.08 175.14 3.49 126.48 187.32 120.5 2.99
Sol_RF 23 75.56 -41.35 180.00 138.65 -151.12 41.35 119.8 2.97
Sol_RF 24 66.12 36.35 180.00 36.35 132.24 143.65 116.6 2.89
Sol_RF 25 83.87 71.62 180.00 71.62 167.74 108.38 114.7 2.85
Sol_RF 26 69.24 -12.37 180.00 167.63 -138.48 12.37 112.3 2.79
Sol_RF 27 59.75 15.26 172.29 7.64 119.07 157.12 112.2 2.78
Sol_RF 28 120.25 -164.74 172.29 22.88 119.07 172.36 112.2 2.78
Sol_RF 29 96.68 -70.99 180.00 109.01 -166.63 70.99 110.9 2.75
Sol_RF 30 63.23 -44.73 180.00 135.27 -126.47 44.73 108.9 2.70
Cheers,
Paul Holland
--
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Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
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D-81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail: kostr...@genzentrum.lmu.de
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