[ccp4bb] Space group and R/Rfree value
Dear all, I am working on a data-set (2.3A) and the space group problem bothers me a lot.The space group of the data-set could be C2221 or P212121, since our protein functions as a dimer, and P212121 gives two molecular in the asym-uint, I think P212121 is more reasonable than C2221.However with C2221, I can refine the R/Rfree to 20/24 or lower, while with P212121 only to 26/30. Also Phenix points out that the crystal is probably a twin with P212121 but is OK with C2221. I am not a real crystallographer, perhaps this problem is stupid, any help will be appreciated!! Best wishes, Xiaopeng Hu
[ccp4bb] [Fwd: Re: [ccp4bb] Space group and R/Rfree value]
Original Message Subject:Re: [ccp4bb] Space group and R/Rfree value Date: Wed, 01 Dec 2010 09:44:06 -0700 From: Maia Cherney ch...@ualberta.ca To: Xiaopeng Hu huxp...@mail.sysu.edu.cn References: 643947201.129232.1291191478190.javamail.r...@zmbx0.sysu.edu.cn A dimer could be with a symmetry-related molecule in C2221, so there is no need to have a dimer in the asym. unit. Maia Xiaopeng Hu wrote: Dear all, I am working on a data-set (2.3A) and the space group problem bothers me a lot.The space group of the data-set could be C2221 or P212121, since our protein functions as a dimer, and P212121 gives two molecular in the asym-uint, I think P212121 is more reasonable than C2221.However with C2221, I can refine the R/Rfree to 20/24 or lower, while with P212121 only to 26/30. Also Phenix points out that the crystal is probably a twin with P212121 but is OK with C2221. I am not a real crystallographer, perhaps this problem is stupid, any help will be appreciated!! Best wishes, Xiaopeng Hu
Re: [ccp4bb] Space group and R/Rfree value
It is not at all unusual for a biological homodimer to sit on a crystallographic two-fold symmetry axis. It is also not unusual for such a dimer to sit entirely in the asymmetric unit. This cannot be used to identify the space group. The space group is determined by the diffraction data. The difference between C2221 and P212121 is that many of the reflections predicted for P212121 have intensity equal to zero in C2221. Since you have a confusion between these two, I presume the P212121 model has a pseudotranslational symmetry of (0.5,0.5,0.0). This pseudo- translational symmetry should be reported by xtriage, and will mislead the twin detection tests. To determine which of these choices is the correct space group you do not perform refinement, you look at the diffraction pattern to see if there are non-zero intensities for the spots that must be zero if the space group were C2221. In P212121 with pseudo-C centering these spots will be weak but observable. I am not surprised that your refinement in P212121 gives higher R values than C2221. In P212121 with pseudo-C symmetry half of the reflections are weak and will have low signal/noise ratio. With the assumption of C centering these weak reflections are discarded and the R values will go down. Your goal is not to reduce the R values, but to fit the data. If these reflections have non-zero intensities you must integrate them and add them to your refinement. Dale Tronrud On 12/01/10 08:31, Xiaopeng Hu wrote: Dear Dr. Kelly Daughtry, Thanks for your help. The enzyme I am working on now functions as a dimmer and the active site is located at the interface. In previously published homology structures, there is one dimmer in the ASU and the dimmer has a tight NCS. With C2221, the dimmer formed by symmetry mates fits the homology dimmer very very well. It is hard for me to understand how a enzyme can has such a crystallographic dimmer. I am not good with Phenix, so I only tried xtriage to check the data set. With C2221, the twin test gives a good Z score which is much smaller than the critic 3.5, while with P212121, the Z score is high (10). I didn't go further. The maps look just the same between the two space groups. - 原始邮件 - 发件人: Kelly Daughtry kddau...@bu.edu 收件人: Xiaopeng Hu huxp...@mail.sysu.edu.cn 发送时间: 星期四, 2010年 12 月 02日 上午 12:05:08 主题: Re: [ccp4bb] Space group and R/Rfree value Sorry, I meant with the P212121 refinement. You mentioned that it is probably twinned. Including the twin law in refinement with the P212121 data should help lower your R and Rfree values. If you have already included the twin law in your phenix refinement for the P212121 data, and R and Rfree can not be lowered, I would suggest that your data probably is C2221. Also, the fact that C2221 is not twinned while P212121 is twinned is an indicator to me that C2221 is probably correct as well. I wouldn't exclude C2221 as the real space group for not having the desired dimer. I have had tetrameric proteins crystallize with one mol / ASU, trimers with one mol/ ASU. If you turn on symmetry mates, do you see your intended dimer with the C2221 data? Last question/suggestion: Do the maps look the same between the two space groups? I would assume that the P212121 calculated maps are somewhat worse than the C2221 maps. With space group identity problems like these, you have to let the data tell you what is the correct space group. And from the looks of it, C2221 is the way to go. Best of luck, Kelly *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** 2010/12/1 Xiaopeng Hu huxp...@mail.sysu.edu.cn 1: No, the data reduction software didnt find twin and C2221 works well, so I never tried twin in refinement. 2: C2221 gives out a monomer in the ASU. - 原始邮件 - 发件人: Kelly Daughtry kddau...@bu.edu 收件人: Xiaopeng Hu huxp...@mail.sysu.edu.cn 发送时间: 星期三, 2010年 12 月 01日 下午 11:32:42 主题: Re: [ccp4bb] Space group and R/Rfree value Just to clarify, did you use the twin law in the phenix refinement? Also, is the C2221 solution a monomer or dimer in the ASU? *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** 2010/12/1 Xiaopeng Hu huxp...@mail.sysu.edu.cn Dear all, I am working on a data-set (2.3A) and the space group problem bothers me a lot.The space group of the data-set could be C2221 or P212121, since our protein functions
Re: [ccp4bb] Space group and R/Rfree value
Hi I'm not sure if anyone has mentioned this yet, but if you use hklview to view the mtz file (assuming you're working in ccp4- land), you can probably get a better handle on the systematic absences than by looking at the original images. I'm told there are other tools available to examine your reflection file graphically, but I haven't used them so I can't comment on them. On 1 Dec 2010, at 17:58, Phoebe Rice wrote: Its may be an interesting question of pseudosymmetry. The best thing would be to find a local mentor who could have a good hands- on look, but here are some thoughts: From the space group tables you will see that P212121 has half as many asymmetric units as C2221, and C2221 has crystallographic twofold axes that your dimer could lie on. So both space groups are perfectly consistent with the protein being a dimer. (You must be able to see the symmetric dimer when you rebuild your model in C2221 during the refinement process?) The R-factor might be higher in P212121 because you're refining two molecules instead of one, and thus there are more degrees of freedom. If the space group does turn out to be P212121, you should probably find a local expert for detailed refinement advice. The centering in C2221 should give a different pattern of systematically missing spots, so you should have a carefull look at the original images and the output of the scaling in C2221, and see if the additional missing spots are really missing or not. (Don't forget the axes may be permuted between the two indexing systems). If they're present but weak, its P212121. In that case, you should note that the systematically weak spots will distort the standard twinning statistics. Hope that helps! Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/ 01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 1 Dec 2010 16:17:58 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Xiaopeng Hu huxp...@mail.sysu.edu.cn) Subject: [ccp4bb] Space group and R/Rfree value To: CCP4BB@JISCMAIL.AC.UK Dear all, I am working on a data-set (2.3A) and the space group problem bothers me a lot.The space group of the data-set could be C2221 or P212121, since our protein functions as a dimer, and P212121 gives two molecular in the asym-uint, I think P212121 is more reasonable than C2221.However with C2221, I can refine the R/Rfree to 20/24 or lower, while with P212121 only to 26/30. Also Phenix points out that the crystal is probably a twin with P212121 but is OK with C2221. I am not a real crystallographer, perhaps this problem is stupid, any help will be appreciated!! Best wishes, Xiaopeng Hu Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] Space group and R/Rfree value
Wouldnt it be easier (in case anyone didnt suggest it yet) to look at the parity analysis from e.g. dataman or probably phenix.xtriage to tell you if you have pseudosymmetry (if you processed it in P212121) -- in C1 you obvioulsy already assume _real_ centering. visually inspecting maybe bit tedious unless you are really hard core on it... Tommi On Dec 1, 2010, at 9:25 PM, harry powell wrote: Hi I'm not sure if anyone has mentioned this yet, but if you use hklview to view the mtz file (assuming you're working in ccp4- land), you can probably get a better handle on the systematic absences than by looking at the original images. I'm told there are other tools available to examine your reflection file graphically, but I haven't used them so I can't comment on them. On 1 Dec 2010, at 17:58, Phoebe Rice wrote: Its may be an interesting question of pseudosymmetry. The best thing would be to find a local mentor who could have a good hands- on look, but here are some thoughts: From the space group tables you will see that P212121 has half as many asymmetric units as C2221, and C2221 has crystallographic twofold axes that your dimer could lie on. So both space groups are perfectly consistent with the protein being a dimer. (You must be able to see the symmetric dimer when you rebuild your model in C2221 during the refinement process?) The R-factor might be higher in P212121 because you're refining two molecules instead of one, and thus there are more degrees of freedom. If the space group does turn out to be P212121, you should probably find a local expert for detailed refinement advice. The centering in C2221 should give a different pattern of systematically missing spots, so you should have a carefull look at the original images and the output of the scaling in C2221, and see if the additional missing spots are really missing or not. (Don't forget the axes may be permuted between the two indexing systems). If they're present but weak, its P212121. In that case, you should note that the systematically weak spots will distort the standard twinning statistics. Hope that helps! Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 1 Dec 2010 16:17:58 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Xiaopeng Hu huxp...@mail.sysu.edu.cn) Subject: [ccp4bb] Space group and R/Rfree value To: CCP4BB@JISCMAIL.AC.UK Dear all, I am working on a data-set (2.3A) and the space group problem bothers me a lot.The space group of the data-set could be C2221 or P212121, since our protein functions as a dimer, and P212121 gives two molecular in the asym-uint, I think P212121 is more reasonable than C2221.However with C2221, I can refine the R/Rfree to 20/24 or lower, while with P212121 only to 26/30. Also Phenix points out that the crystal is probably a twin with P212121 but is OK with C2221. I am not a real crystallographer, perhaps this problem is stupid, any help will be appreciated!! Best wishes, Xiaopeng Hu Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
