Re: [ccp4bb] Molecular replacement question

[Roger Rowlett]

Here is how I would approach this: Use Phaser to search with monomers, or dimers if you suspect that the biological unit is composed of dimers and have reasonable guess at a dimer search model. Also consider searching with N- and C-terminal truncated search models. Sometimes a wayward N- or C-terminal helix or loop makes a solution impossible to find because of packing issues. No joy? Don't give up yet, try EPMR. It is especially good at finding multiple chains in the ASU. If searching for multiple copies of protein in the ASU, set the search criterion for CC=1 to force an exhaustive search for partial solutions. (With the default setting, EPMR gives up too early in our experience). We have found that in difficult cases, you can use some or all of your high-resolution data to improve the search in EPMR. (It is slow as all get-out when you do this, but sometimes worth the wait. It's only CPU cycles...) The resolution range you use in EPMR can have a profound impact on the ability to find a solution. Examine any and all candidate solutions for packing in the ASU. You may have to relax the packing criteria for Phaser and EPMR to allow the solution to be found. If any of these look like they pack reasonably well, they *might* be good starting points for density modification. Partial solutions may give you some hints about how many protein units you need in the ASU. We've had good luck in a recent "hard nut to crack" using a marginal Phaser solution that packed well, followed by DM using Parrot and autobuilding with Buccaneer. The basic orientation of the proteins in the ASU were properly predicted by Phaser (8 monomers or 4 dimers--only the dimer search worked), but the initial map was uninterpretable before DM. I think the rms difference between the search model and final solution in this case was about 1.5 A. Cheers. Paul Holland wrote: 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 -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu