Sorry for this long message... We've been working with data from a crystal form of an unligated protein (140 residues) that diffracts to 2.8 Å. The data index as P3(i)21 with an Rmerge of 4.6% overall (vs. 4.4% in P3). The unit cell dimensions are 42x42x149, which is consistent with single subunit in the asymmetric unit of P3(i)21. The protein is active as a homodimer, and as expected the best molecular replacement hits put the molecular two-fold along the crystallographic two-fold. P3(2)21 does not give as good results. (The search model is the same protein molecule bound to its ligand). When rigid body refinement (to 3.5 Å) is performed on this molecular replacement solution, the R/Rfree is in the 35% range, but simulated annealing (with data to 2.8 Å) causes immediate problems, with the working R dropping to about 32% as the Rfree goes to above 40%. Electron density maps are poor throughout the model, and efforts at rebuilding have not improved things. I have tried to use other space groups, including P3(1) and C2 (dimensions 73 x 42 x 149, 90 x 90.1 x 90) in hopes of reducing symmetry constraints on the refinement, but the problem remains. An obvious issue that sprung to mind is twinning, but I saw no evidence of twinning when I use Todd Yeates' server, the CNS & CCP4 utilities, or when I look at intensity distribution in the Truncate plot. A recent thread on this bulletin board described a problem similar to mine (my thanks to Michael Hothorn and the several responders). Folks recommended using the Padilla-Yeates analysis in dataman. I've done that now, and have come up with an odd result that I don't understand - the data (either integrated as P3 or P321) fall well below the curve expected for either twinned or untwinned data, with <|L|> = 0.592 and <L^2>=0.439. The plot from dataman is available at: If someone can help interpret this plot I'd be grateful. The self-rotation maps and native pattersons don't reveal anything that I can readily interpret, though there are some weak peaks in both that might have meaning and I'm just oblivious to their importance. The self-rotation map (from GLRF) has two-folds where it ought (at around 11 sigma) and several weaker peaks (around 4 sigma): The peaks above 5 sigma in the native patterson are listed below (calc'd in CNS) and a link to the Harker section is given below as well. peak no. interpolated (x,y,z,height) grid point (x,y,z,rho) (fractional coordinates) 1 0.0667 0.0000 0.0000 8.3871 0.0667 0.0000 0.0000 8.3871 2 0.6667 0.3333 0.9891 7.2202 0.6667 0.3333 0.9867 6.8977 3 0.5520 0.1026 0.9059 6.0885 0.5556 0.1111 0.9067 6.0000 4 0.6667 0.3333 0.7735 5.7798 0.6667 0.3333 0.7733 5.7790 5 0.0000 0.0000 0.0662 5.7358 0.0000 0.0000 0.0667 5.7241 6 0.0000 0.0000 0.0344 5.0269 0.0000 0.0000 0.0333 4.9362 I've read that having a molecular two-fold aligned with a crystallographic axis can make twinning difficult to detect, but given the above results I'm not sure if that's really what's going on, nor how to work around the potential problem. I feel like I must be missing something obvious, but after several weeks of struggling, I'm hoping someone can enlighten me. Thanks in advance, Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA |
- [ccp4bb]: refinement problem Arthur Glasfeld
- Re: [ccp4bb]: refinement problem William Scott
