unusual maps and Rfree for 1.75 structure
I am solving a structure of an enzyme that I have solved many times before, the only difference is a single mutation in the active site. The crystal grew in basically exact conditions (macroscopically speaking they look different) as the other mutants I had. I scaled the data P 43 21 2 Rsym 0.068. Like all the other data sets I had before. This time however, the initial model has Rfree of about 0.27 and the maps look more like those of a 2.5 - 2.8 structure. My experinence with this enzyme is that I should be looking at around 0.22 or lower Rfree, and pretty clear maps as I saw for my 1.9 structure. Xtriage does not detect any pathologies. I rececked my scaling log file, and it lists the systematic absences for the data. I see some that are not that "weak" for instance : h k l I/sigma 0 0 1 6.0 0 0 2 5.8 0 0 13 7.8 So I am inclined to reprocess my data in P 4 2 2. Are these "symptoms" of a possible wrong space group assignment? Is there something else obvious I am overlooking? Thank you -- Yuri Pompeu
On Sat, Apr 16, 2011 at 7:50 PM, Yuri
So I am inclined to reprocess my data in P 4 2 2. Are these "symptoms" of a possible wrong space group assignment? Is there something else obvious I am overlooking?
I don't know enough to comment on the systematic absences, but if Xtriage doesn't indicate any problems with the resolution cutoff, severe anisotropy, twinning, etc., my suggestion would be to look at the images to make sure you indexed them correctly. The command 'labelit.index' is your friend here - it will look for evidence of pseudotranslation in the diffraction patterns, which the integration software tends to overlook. Ignoring the weak spots in the images will usually still result in good scaling statistics and easy MR solutions, and probably won't trigger any warnings in Xtriage, but it will result in abnormally high R-factors. (I'm not sure how common this type of error is, but I once wasted several months trying to refine against incorrectly processed data, so it's always the first thing I suggest looking at.) (By the way, if you're using the Xtriage GUI, the summary tab may be a little misleading - interpreting the raw numbers is very subjective, and I tried not to make the feedback too alarmist. So the absence of obvious warnings may not mean that everything is okay. If you send the Xtriage log file to [email protected] we may be able to make a more definitive judgement.) -Nat
Hi Yuri, you did not mention the resolution... From your email I guess it is 1.9A, is it correct? Anyway, to bypass the uncertainties of the space group choice (and artifacts due to constraints it applies) you can always process your data in P1, solve and refine you model (applying NCS if necessary; and yes, you are not solving completely new structure - you solved it many times before, so you know the answer pretty much!) and see how the solution obtained this way compares with whatever else you get from more wise strategy. Pavel. On 4/16/11 7:50 PM, Yuri wrote:
I am solving a structure of an enzyme that I have solved many times before, the only difference is a single mutation in the active site. The crystal grew in basically exact conditions (macroscopically speaking they look different) as the other mutants I had. I scaled the data P 43 21 2 Rsym 0.068. Like all the other data sets I had before. This time however, the initial model has Rfree of about 0.27 and the maps look more like those of a 2.5 - 2.8 structure. My experinence with this enzyme is that I should be looking at around 0.22 or lower Rfree, and pretty clear maps as I saw for my 1.9 structure. Xtriage does not detect any pathologies. I rececked my scaling log file, and it lists the systematic absences for the data. I see some that are not that "weak" for instance : h k l I/sigma 0 0 1 6.0 0 0 2 5.8 0 0 13 7.8
So I am inclined to reprocess my data in P 4 2 2. Are these "symptoms" of a possible wrong space group assignment? Is there something else obvious I am overlooking? Thank you
-- Yuri Pompeu
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participants (3)
-
Nathaniel Echols -
Pavel Afonine -
Yuri