Re: [ccp4bb] Pseudo-translation problem
In terms of detecting pseudo translational symmetry, it sounds like both crystals have it, but the relationship between the two complexes is closer to perfect in the 2nd crystal. Looking at that problematic density, it does indeed suggest that the lattice is slightly messed up. For instance, you could have a sort of twinning-ish problem. Say, if your molecules usually sit in a row like such: Mol1 - x Angstroms - Mol2 - 0.9x Angstroms - Mol1 - etc. but some rows of the crystal pack: Mol1 - 0.9x Angstroms - Mol2 - x Angstroms - Mol1 etc, the Mol1s will superimpose but the Mol2s won't. Or could it be that sometimes the protein binds 1bp over from where you wanted it to? Do you have any halogens on the DNA that you could use to make sure the DNA sequence register is always the same (i.e. if you put in 1 bromo-dU, do you get 1 anomalous peak or two)? Since you don't have a problem with the density derived from your first data set, you might get lucky just by cranking through many different crystals of the 2nd complex. I might try micro seeding as well in hopes of somehow tweaking the crystal growth pattern. As a side point, a Patterson peak that is 18% of the origin may not be huge, but it seems unusually large to me. I suspect there's a broad grey area where you need some additional analysis to decide what is really noteworthy pseudo translational symmetry, such as noticeable patterns of dark vs. light spots. As a "control", I looked up the validation report now posted for one of my lab's old structures that had pseudo translational symmetry clearly visible in the diffraction pattern (and later in the coordinates) (1szp). Xtriage found a 19%-of-origin peak for that, and decided "no signifiant pseudo translation." Phoebe ++ Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu<mailto:pr...@uchicago.edu> http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Minyun Zhou [minyunz...@gmail.com] Sent: Wednesday, July 30, 2014 9:58 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Pseudo-translation problem Dear all, I am trying to determine the structure of a protein-DNA complex. I collected several datasets of the same protein in complex with two dsDNAs. Two DNAs have the same sequence, but one DNA contain a single central mispair, which is cleaved by the enzyme leaving an abasic site, while the other contains a non-cleavable mispair. Two datasets have almost the same space group and cell parameter, however, the later one has been detected with pseudo-translation while the other not. The details of two datasets are as follows: Dataset 1. Protein with abasic site containing dsDNA: C2221: a=106, b=111, c=182.2, α=β=γ=90 Resolution: 2.45 A Xtriage summary: The largest off-origin peak in the Patterson function is 18.57% of the height of the origin peak. No significant pseudotranslation is detected. Dataset 2. Protein with non-cleavable lesion containing dsDNA: C2221: a=106.1, b=111.1, c=183, α=β=γ=90 Resolution: 3.0 A Xtriage summary: The analysis of the Patterson function reveals a significant off-origin peak that is 60.75 % of the origin peak, indicating pseudo translational symmetry. I first determined the structure using the dataset 1 by MR. There are two protein-DNA complexes in one asymmetric unit, and two molecules are similar except the conformation of a small loop at the active site. The final model has the Rwork/Rfree of 19.3%/23.0%. Then I use this refined structure as model to solve the second structure with dataset 2. The final Rwork/Rfree is 24.3%/28.9%. The DNA density looks okay while the protein part is problematic. After refinement, although the main chain of the protein is fitted well into the density, there is still a lot of unidentified continuous positive density next to the polypeptide chain, especially near the region involved in the crystal packing. I attached a snapshot below, which shows one of these positive densities lies along a helix. Since there is no other polymer in the reservoir solution that can fit, the additional density seems to indicate another slightly shifted conformation of the protein itself. My question is whether this seemingly “dual conformation” is caused by pseudo-translation and indicates the solution I found is incorrect. And how can I eliminate the effect of pseudo-translation to get the correct structure? Thanks for your help! Best regards, Minyun
Re: [ccp4bb] Pseudo-translation problem
Hi, Lattice-translocation disorder is one possibility. How does the diffraction image look like? Are there any streaks? Best regards, Takanori Nakane On 2014-07-30 15:58, Minyun Zhou wrote: Dear all, I am trying to determine the structure of a protein-DNA complex. I collected several datasets of the same protein in complex with two dsDNAs. Two DNAs have the same sequence, but one DNA contain a single central mispair, which is cleaved by the enzyme leaving an abasic site, while the other contains a non-cleavable mispair. Two datasets have almost the same space group and cell parameter, however, the later one has been detected with pseudo-translation while the other not. The details of two datasets are as follows: Dataset 1. Protein with abasic site containing dsDNA: C2221: a=106, b=111, c=182.2, α=β=γ=90 Resolution: 2.45 A Xtriage summary: The largest off-origin peak in the Patterson function is 18.57% of the height of the origin peak. No significant pseudotranslation is detected. Dataset 2. Protein with non-cleavable lesion containing dsDNA: C2221: a=106.1, b=111.1, c=183, α=β=γ=90 Resolution: 3.0 A Xtriage summary: The analysis of the Patterson function reveals a significant off-origin peak that is 60.75 % of the origin peak, indicating pseudo translational symmetry. I first determined the structure using the dataset 1 by MR. There are two protein-DNA complexes in one asymmetric unit, and two molecules are similar except the conformation of a small loop at the active site. The final model has the Rwork/Rfree of 19.3%/23.0%. Then I use this refined structure as model to solve the second structure with dataset 2. The final Rwork/Rfree is 24.3%/28.9%. The DNA density looks okay while the protein part is problematic. After refinement, although the main chain of the protein is fitted well into the density, there is still a lot of unidentified continuous positive density next to the polypeptide chain, especially near the region involved in the crystal packing. I attached a snapshot below, which shows one of these positive densities lies along a helix. Since there is no other polymer in the reservoir solution that can fit, the additional density seems to indicate another slightly shifted conformation of the protein itself. My question is whether this seemingly “dual conformation” is caused by pseudo-translation and indicates the solution I found is incorrect. And how can I eliminate the effect of pseudo-translation to get the correct structure? Thanks for your help! Best regards, Minyun
Re: [ccp4bb] Pseudo-translation?
The general rule is that a Patterson peak should be ~ 20% of the origin before considering it significant so none of those would really need to be considered. Eleanor On 25 February 2013 12:48, Michele Lunelli wrote: > Dear all, > > I have an orthorhombic crystal (pointless suggests most likely space > groups P 2 21 21 or P 2 21 2) with two molecules expected in the asymmetric > unit. Analyzing the native Patterson map I found the following peaks (in > fractional coordinates): > > CELL 63.0400 117.2500 133.6500 90. 90. 90. > ATOM1 Ano 0. 0. 0. 111.03 0.0 BFAC 20.0 > ATOM2 Ano 0.0102 0.0416 0. 15.98 0.0 BFAC 20.0 > ATOM3 Ano 0. 0.5000 0.00376.21 0.0 BFAC 20.0 > ATOM4 Ano 0.0669 0.0850 0.5.86 0.0 BFAC 20.0 > ATOM5 Ano 0.0772 0.4168 0.5.78 0.0 BFAC 20.0 > ATOM6 Ano 0.1139 0.1196 0.00495.10 0.0 BFAC 20.0 > > The third and fifth are far enough from the origin to represent > tranlations. Is the third due to an alternative origin? Could the fifth > represent a pseudo-translation? > > > Thanks, > Michele >
[ccp4bb] Pseudo-translation?
Dear all, I have an orthorhombic crystal (pointless suggests most likely space groups P 2 21 21 or P 2 21 2) with two molecules expected in the asymmetric unit. Analyzing the native Patterson map I found the following peaks (in fractional coordinates): CELL 63.0400 117.2500 133.6500 90. 90. 90. ATOM1 Ano 0. 0. 0. 111.03 0.0 BFAC 20.0 ATOM2 Ano 0.0102 0.0416 0. 15.98 0.0 BFAC 20.0 ATOM3 Ano 0. 0.5000 0.00376.21 0.0 BFAC 20.0 ATOM4 Ano 0.0669 0.0850 0.5.86 0.0 BFAC 20.0 ATOM5 Ano 0.0772 0.4168 0.5.78 0.0 BFAC 20.0 ATOM6 Ano 0.1139 0.1196 0.00495.10 0.0 BFAC 20.0 The third and fifth are far enough from the origin to represent tranlations. Is the third due to an alternative origin? Could the fifth represent a pseudo-translation? Thanks, Michele
[ccp4bb] pseudo translation
Hi all,here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K
[ccp4bb] pseudo-translation vectors in molrep vs other programs
Dear colleagues, I would like to thank J. Murray, J. Wright, K. Futterer, E. Dodson, A. Forster, and F. Long for responding to my posting of two days ago on pseudo-translation vectors in molrep vs other programs (see original posting at the end of this message). I should have said at the outset that we are dealing with a limiting data set (see stats below), but since this is the only data we were ever able to collect on this membrane protein, we have no option but to milk it as much as we can. P21 with 104.82 151.28 109.49 90.00 118.13 90.00 Resolution: 30-4.2 angs (4.3-4.2) Rmeas=0.15 (0.380) I/sigma: 7.2 (1.9) Completeness=93% (75%) Redundancy= 2.3 (2.1) Mosaicity= 1.1 deg High data anisotropy, primarily along the K reciprocal axis. The comments from Eleanor Dodson and Klaus Futterer prompted me to take another look at the data frame per frame. I concluded that in several frames there were a few reflections in the 40-30 angs range that obviously did not fit my spot-integration strategy very well. After failing repeatedly to get them to integrate acceptably without compromising the rest of the data too much, I decided to exclude all reflections between 40 and 30 angs res. This has resulted in three important improvements: (1) Better data integration and scaling statistics across the board. (1) The spurious peaks clustering around the origin in the native patterson are fewer, and those that do remain have a peak-height around 10-12% of the origin. (2) The new data set has yielded unambiguous peaks in the self-rotation function consistent with a 2-fold NCS axis. I have now used this SRF peak in MolRep and came up with a reasonable MR solution. I will soon try to implement this SRF info in PHASER as well via the "Rotate around" option. Best regards Savvas Savvas N. Savvides Unit for Structural Biology and Biophysics Laboratory for Protein Biochemistry - Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19 Email: [EMAIL PROTECTED] http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html > > Dear colleagues, > > > > For a particular MR problem I am dealing with, 'analyse_mr' suggests > > that there maybe a pseudo-translation vector as evidenced by the very > > significant non-origin peaks in the native patterson: e.g > > > > GRID 80 112 80 > > CELL 104.8290 151.2840 109.4910 90. 118.1310 90. > > ATOM1 Ano 0. 0. 0. 181.08 0.0 BFAC 20.0 > > ATOM2 Ano 0.9483 0. 0.0106 46.89 0.0 BFAC 20.0 > > ATOM3 Ano 0.0517 0. 0.9875 46.89 0.0 BFAC 20.0 > > ATOM4 Ano 0.9494 0.9911 0.0090 40.66 0.0 BFAC 20.0 > > ATOM5 Ano 0.0506 0.9911 0.9875 40.66 0.0 BFAC 20.0 > > ATOM6 Ano 0.0572 0.9911 0. 37.26 0.0 BFAC 20.0 > > > > BALBES also reports a pseudo-translation vector at 0.951 0.000 0.007, > > i.e. very similar to the output from 'analyse_mr'. > > > > Yet, Molrep fails to recognize this possibility (in "auto' mode for > > the PST) claiming that the 0.125 limit for the peak height compared to > > the origin has not been reached. When I look at the output from > > 'analyse_mr' it is quite clear the peak is at 0.25 of the origin peak. > > > > Why is there such a discrepancy in the interpretation of the native > > patterson map? > > > > Best regards > > Savvas
Re: [ccp4bb] pseudo-translation vector in molrep
Hi, I suggest you check which version of MOLREP you used. Currently, BALBES now actually use MOLREP in 'auto' mode for PST. The two should be the same. The difference may be because BALBES uses the latest version of MOLREP. Fei On 7/30/07, Eleanor Dodson <[EMAIL PROTECTED]> wrote: > I think the error is in BALBES - there is a peak I guess at 0.95 0 0.01 > but it must be too close to the origin to be a translation vector from > one molecule to another. > > There are reasons for such peaks - sometimes spurious large terms in the > data.. > but they dont usually represent true molecular translations. > > Trust MOLREP! > > Eleanor > > Savvas Savvides wrote: > > > > Dear colleagues, > > > > For a particular MR problem I am dealing with, 'analyse_mr' suggests > > that there maybe a pseudo-translation vector as evidenced by the very > > significant non-origin peaks in the native patterson: e.g > > > > GRID 80 112 80 > > CELL 104.8290 151.2840 109.4910 90. 118.1310 90. > > ATOM1 Ano 0. 0. 0. 181.08 0.0 BFAC 20.0 > > ATOM2 Ano 0.9483 0. 0.0106 46.89 0.0 BFAC 20.0 > > ATOM3 Ano 0.0517 0. 0.9875 46.89 0.0 BFAC 20.0 > > ATOM4 Ano 0.9494 0.9911 0.0090 40.66 0.0 BFAC 20.0 > > ATOM5 Ano 0.0506 0.9911 0.9875 40.66 0.0 BFAC 20.0 > > ATOM6 Ano 0.0572 0.9911 0. 37.26 0.0 BFAC 20.0 > > > > BALBES also reports a pseudo-translation vector at 0.951 0.000 0.007, > > i.e. very similar to the output from 'analyse_mr'. > > > > Yet, Molrep fails to recognize this possibility (in "auto' mode for > > the PST) claiming that the 0.125 limit for the peak height compared to > > the origin has not been reached. When I look at the output from > > 'analyse_mr' it is quite clear the peak is at 0.25 of the origin peak. > > > > Why is there such a discrepancy in the interpretation of the native > > patterson map? > > > > Best regards > > Savvas > > > > > > > > Savvas N. Savvides > > [EMAIL PROTECTED] for Structural Biology and Biophysics > > Laboratory for Protein Biochemistry - Ghent University > > K.L. Ledeganckstraat 35 > > 9000 Ghent, BELGIUM > > Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19 > > Email: [EMAIL PROTECTED] > > _http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html_ > > > > >
Re: [ccp4bb] pseudo-translation vector in molrep
I think the error is in BALBES - there is a peak I guess at 0.95 0 0.01 but it must be too close to the origin to be a translation vector from one molecule to another. There are reasons for such peaks - sometimes spurious large terms in the data.. but they dont usually represent true molecular translations. Trust MOLREP! Eleanor Savvas Savvides wrote: Dear colleagues, For a particular MR problem I am dealing with, 'analyse_mr' suggests that there maybe a pseudo-translation vector as evidenced by the very significant non-origin peaks in the native patterson: e.g GRID 80 112 80 CELL 104.8290 151.2840 109.4910 90. 118.1310 90. ATOM1 Ano 0. 0. 0. 181.08 0.0 BFAC 20.0 ATOM2 Ano 0.9483 0. 0.0106 46.89 0.0 BFAC 20.0 ATOM3 Ano 0.0517 0. 0.9875 46.89 0.0 BFAC 20.0 ATOM4 Ano 0.9494 0.9911 0.0090 40.66 0.0 BFAC 20.0 ATOM5 Ano 0.0506 0.9911 0.9875 40.66 0.0 BFAC 20.0 ATOM6 Ano 0.0572 0.9911 0. 37.26 0.0 BFAC 20.0 BALBES also reports a pseudo-translation vector at 0.951 0.000 0.007, i.e. very similar to the output from 'analyse_mr'. Yet, Molrep fails to recognize this possibility (in "auto' mode for the PST) claiming that the 0.125 limit for the peak height compared to the origin has not been reached. When I look at the output from 'analyse_mr' it is quite clear the peak is at 0.25 of the origin peak. Why is there such a discrepancy in the interpretation of the native patterson map? Best regards Savvas Savvas N. Savvides [EMAIL PROTECTED] for Structural Biology and Biophysics Laboratory for Protein Biochemistry - Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19 Email: [EMAIL PROTECTED] _http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html_
[ccp4bb] pseudo-translation vector in molrep
Dear colleagues, For a particular MR problem I am dealing with, 'analyse_mr' suggests that there maybe a pseudo-translation vector as evidenced by the very significant non-origin peaks in the native patterson: e.g GRID 80 112 80 CELL 104.8290 151.2840 109.4910 90. 118.1310 90. ATOM1 Ano 0. 0. 0. 181.08 0.0 BFAC 20.0 ATOM2 Ano 0.9483 0. 0.0106 46.89 0.0 BFAC 20.0 ATOM3 Ano 0.0517 0. 0.9875 46.89 0.0 BFAC 20.0 ATOM4 Ano 0.9494 0.9911 0.0090 40.66 0.0 BFAC 20.0 ATOM5 Ano 0.0506 0.9911 0.9875 40.66 0.0 BFAC 20.0 ATOM6 Ano 0.0572 0.9911 0. 37.26 0.0 BFAC 20.0 BALBES also reports a pseudo-translation vector at 0.951 0.000 0.007, i.e. very similar to the output from 'analyse_mr'. Yet, Molrep fails to recognize this possibility (in "auto' mode for the PST) claiming that the 0.125 limit for the peak height compared to the origin has not been reached. When I look at the output from 'analyse_mr' it is quite clear the peak is at 0.25 of the origin peak. Why is there such a discrepancy in the interpretation of the native patterson map? Best regards Savvas Savvas N. Savvides [EMAIL PROTECTED] for Structural Biology and Biophysics Laboratory for Protein Biochemistry - Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19 Email: [EMAIL PROTECTED] http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html
