Re: [ccp4bb] alternating strong/weak intensities in reciprocal planes - P622
If you have a high off origin peak at 0 0 0.5 you must have absences along the 00l axis also consistent with P63 22. So you need to test both P6322 and P6 22 And is the tetramer generated by crystal symmetry or is the whole thing in the asymmetric unit? Eleanor Jorge Iulek wrote: Dear all, Please, maybe you could give some suggestions to the problem below. 1) Images show smeared spots, but xds did a good job integrating them. The cell is 229, 229, 72, trigonal, and we see alternating strong and weak rows of spots in the images (spots near each other, but rows more separated, must be by c*). They were scaled with xscale, P622 (no systematic abscences), R_symm = 5.3 (15.1), I/sigI = 34 (14) and redundancy = 7.3 (6.8), resolution 2.8 A. Reciprocal space show strong spots at h, k, l=2n and weak spots at h, k, l=2n+1 (I mean, l=2n intensities are practically all higher than l=2n+1 intensities, as expected from visual inspection of the images). Within planes h, k, l=2n+1, the average intensity is clearly and much *higher at high resolution than at low resolution*. Also, within planes h, k, l=2n, a subjective observation is that average intensity apparently does not decay much from low to high resolution. The data were trucated with truncate, which calculated Wilson B factor to be 35 A**2. 2) Xtriage points a high (66 % of the origin) off-origin Patterson peak. Also, ML estimate of overall B value of F,SIGF = 25.26 A**2. 3) I suspect to have a 2-fold NCS parallel to a (or b), halfway the c parameter, which is almost crystallographic. 4) I submitted the data to the Balbes server which using pseudo-translational symmetry suggested some solutions, one with a good contrast to others, with a 222 tetramer, built from a structure with 40 % identity and 58% positives, of a well conserved fold. 5) I cannot refine below 49 % with either refmac5, phenix.refine or CNS. Maps are messy, except for rather few residues and short stretches near the active site, almost impossible for rebuilding from thereby. Strange, to me, is that all programs freeze all B-factors, taking them the program minimum (CNS lowers to almost its minimum). Might this be due to by what I observed in the reciprocal space as related in 1 ? If so, might my (intensity) scaling procedure have messed the intensities due to their intrinsic property to be stronger in alternating planes ? How to overcome this ? 6) I tried some different scaling strategies *in the refinement step*, no success at all. 7) A Patterson of the solution from Balbes also shows an off-origin Patteron at the same position of the native data, although a little lower. 8) Processed in P6, P312 and P321, all of course suggest twinning. I would thank suggestions, point to similar cases, etc... In fact, currently I wondered why refinement programs take B-factor to such low values Many thanks, Jorge
[ccp4bb] alternating strong/weak intensities in reciprocal planes - P622 - feedback / quasi SUMMARY
I got several suggestions. I thought it is time to answer some questions, prepare a partial quasi- summary and give some more feedback. Please, keep sending me any more insights and suggestions you might have. Briefly, the problem is: P622 (no systematic abscences), strong spots at h, k, l=2n and weak spots at h, k, l=2n+1; planes h, k, l=2n+1, the average intensity is clearly and much *higher at high resolution than at low resolution*; off-origin Patterson peak; refinement programs freeze all B-factors of a supposed 222 point group MR solution. I had not mentioned that the off-origin Patterson peak is at 0, 0, and 0.5. Josh Waren (JW), Dale Tronrud (DT), Bart Hazes (BH) and Pierre Rizkallah (PR) put their suggestions to the bb, Tommi Kajander (TK), Anastassis Perraskis (AP), Esko Oksaken (EO), Peter Zwart (PZ), Robyn Stanfield (RS), Jose Antonio cuesta-Seijo (JACS), Benajmin Bax (BB) and Xueyong Zhu (XZ) wrote pvt messages, I will include their comments in this summary as this is a general practive. JI below is me (Jorge Iulek). TK and EO: a 2006 paper by EO rigid body refine with weak refl and refine with the rest later. Also, maybe try lower symmetry. Scale weak and strong data separately yet assumption of independent data is no longer valid. (JI): I should read the paper to check how to scale them separately; for while my simple idea is to use rzone in mtzutils before scaling, now, with scala. JW: Check the suggested twinning fractions at the lower sg's, specially a partial twin fraction of near 0.5 _and_ perfect twinning, or if the stats suggest partial twinning with a lower twin fraction. Particular attention to the l statistic. AP: remembers the good processing statistics at the higher resolution, so get more resolution to facilitate things. Possibilities are to FeDEX xtals to APS and ESRF. (JI): this will be on mind, but currently there are several difficulties to express the protein again. The resolution was limited by instrumentation, spot smearing and the 230 A parameters. PZ: read about OD twins, while work with regular twin. Points an 2007 Acta D paper. RS: maybe a lattice translocation defect, check if your weak spots are smeared, and the strong spots are sharp. There a papers decribing this. (JI): Robyn, on eye both strong and weak spots look smeared, but this might be subjective. I would thank you for the complete references. DT: frozen B-factors, ensure your bulk solvent correction is operating correctly. (JI) I tried Babinet's scaling or/and bulk sovent mask with all data and obseved the freeze. I also cut data at 8, 6 and 5 A, no bulk solvent model at all, I got marginally better R's, B factors slightly higher than the floor (under refmac). JACS: had a similar case, due to a NCS 2-fold rotation aligned with a crystallographic 2 fold axis. MR worked equally well ... refinement would stall at terrible R values. ; careful trimming of the initial model .. led to a better behaved refinement ; low B values are due to overfitting He suggests to cut away different smal parts of the structures, see the statistics and the maps if the chuncks show up again. Also, fix Wilson B values. BH: suggests pseudo body centering, off-origin Patterson peak at 0.5, 0.5, 0.5. (JI) Sorry, I added this important information only in this e-mail, it is at 0, 0, 0.5. BH: lower symmetry with twin fraction near 0.5. Refinement may be hard even if the crystal conditions are figured out. Additives might change pseudo to true symmetry. (JI) As pointed above, currently difficulties to get more protein. BH: A wrong model that would obey the pseudo body centering would give good R's and correlations. Trying a whole bunch of rotation function solutions and see which one will refine to a significantly lower R-free is one thing to try (JI) MR was a kind of straight forward with Balbes, I can still check and think more about the results. BB: had a true P21 case which looked hexagonal. Calculated mine to possibly be 114.5, 72, 229, beta=120 degrees, but wonders if this would lead more to a screw 3-fold axis. (JI) Lower symmetry is a possibility I should still look better into. XZ: guessed correctly the off-origin Patterson peak and talks about lattice translocation, with a 2005 Acta D paper as reference. Also, talks about a program for strong pseudo-translation. (JI) Yes, the peak is strong, and I would like to see the program. PR: pacakes of 229x229x36 A slightly shifted in the plane normal to the 6 fold, or slightly rotated out of that plane. Perturbation really small which explain the intensity distribution profile I described. (JI) That is my initial guess. PR: fix a good MR solution and use the same rotation solution (or one very close to it) to find a second translation solution. This should be within a small fraction of 0,0,0.5. After rigid body refinement, you might see the rotation of the 'pancake' clearly. try all the screw ... 6, 6(1), 6(2), 6(3), 6(4) and
[ccp4bb] alternating strong/weak intensities in reciprocal planes - P622
Dear all, Please, maybe you could give some suggestions to the problem below. 1) Images show smeared spots, but xds did a good job integrating them. The cell is 229, 229, 72, trigonal, and we see alternating strong and weak rows of spots in the images (spots near each other, but rows more separated, must be by c*). They were scaled with xscale, P622 (no systematic abscences), R_symm = 5.3 (15.1), I/sigI = 34 (14) and redundancy = 7.3 (6.8), resolution 2.8 A. Reciprocal space show strong spots at h, k, l=2n and weak spots at h, k, l=2n+1 (I mean, l=2n intensities are practically all higher than l=2n+1 intensities, as expected from visual inspection of the images). Within planes h, k, l=2n+1, the average intensity is clearly and much *higher at high resolution than at low resolution*. Also, within planes h, k, l=2n, a subjective observation is that average intensity apparently does not decay much from low to high resolution. The data were trucated with truncate, which calculated Wilson B factor to be 35 A**2. 2) Xtriage points a high (66 % of the origin) off-origin Patterson peak. Also, ML estimate of overall B value of F,SIGF = 25.26 A**2. 3) I suspect to have a 2-fold NCS parallel to a (or b), halfway the c parameter, which is almost crystallographic. 4) I submitted the data to the Balbes server which using pseudo-translational symmetry suggested some solutions, one with a good contrast to others, with a 222 tetramer, built from a structure with 40 % identity and 58% positives, of a well conserved fold. 5) I cannot refine below 49 % with either refmac5, phenix.refine or CNS. Maps are messy, except for rather few residues and short stretches near the active site, almost impossible for rebuilding from thereby. Strange, to me, is that all programs freeze all B-factors, taking them the program minimum (CNS lowers to almost its minimum). Might this be due to by what I observed in the reciprocal space as related in 1 ? If so, might my (intensity) scaling procedure have messed the intensities due to their intrinsic property to be stronger in alternating planes ? How to overcome this ? 6) I tried some different scaling strategies *in the refinement step*, no success at all. 7) A Patterson of the solution from Balbes also shows an off-origin Patteron at the same position of the native data, although a little lower. 8) Processed in P6, P312 and P321, all of course suggest twinning. I would thank suggestions, point to similar cases, etc... In fact, currently I wondered why refinement programs take B-factor to such low values Many thanks, Jorge
Re: [ccp4bb] alternating strong/weak intensities in reciprocal planes - P622
On possibility for #5, the B factors all dropping to the lower limit during refinement. If you are including all of your low resolution data (which you should) but have not used a model for the bulk solvent scattering of X-rays (which would be bad) then you will observe this result. The refinement program will attempt to overestimate the amplitudes of the high resolution Fc's to match the overestimated low resolution Fc's. Check you log files to ensure you bulk solvent correction is operating correctly. Dale Tronrud Jorge Iulek wrote: Dear all, Please, maybe you could give some suggestions to the problem below. 1) Images show smeared spots, but xds did a good job integrating them. The cell is 229, 229, 72, trigonal, and we see alternating strong and weak rows of spots in the images (spots near each other, but rows more separated, must be by c*). They were scaled with xscale, P622 (no systematic abscences), R_symm = 5.3 (15.1), I/sigI = 34 (14) and redundancy = 7.3 (6.8), resolution 2.8 A. Reciprocal space show strong spots at h, k, l=2n and weak spots at h, k, l=2n+1 (I mean, l=2n intensities are practically all higher than l=2n+1 intensities, as expected from visual inspection of the images). Within planes h, k, l=2n+1, the average intensity is clearly and much *higher at high resolution than at low resolution*. Also, within planes h, k, l=2n, a subjective observation is that average intensity apparently does not decay much from low to high resolution. The data were trucated with truncate, which calculated Wilson B factor to be 35 A**2. 2) Xtriage points a high (66 % of the origin) off-origin Patterson peak. Also, ML estimate of overall B value of F,SIGF = 25.26 A**2. 3) I suspect to have a 2-fold NCS parallel to a (or b), halfway the c parameter, which is almost crystallographic. 4) I submitted the data to the Balbes server which using pseudo-translational symmetry suggested some solutions, one with a good contrast to others, with a 222 tetramer, built from a structure with 40 % identity and 58% positives, of a well conserved fold. 5) I cannot refine below 49 % with either refmac5, phenix.refine or CNS. Maps are messy, except for rather few residues and short stretches near the active site, almost impossible for rebuilding from thereby. Strange, to me, is that all programs freeze all B-factors, taking them the program minimum (CNS lowers to almost its minimum). Might this be due to by what I observed in the reciprocal space as related in 1 ? If so, might my (intensity) scaling procedure have messed the intensities due to their intrinsic property to be stronger in alternating planes ? How to overcome this ? 6) I tried some different scaling strategies *in the refinement step*, no success at all. 7) A Patterson of the solution from Balbes also shows an off-origin Patteron at the same position of the native data, although a little lower. 8) Processed in P6, P312 and P321, all of course suggest twinning. I would thank suggestions, point to similar cases, etc... In fact, currently I wondered why refinement programs take B-factor to such low values Many thanks, Jorge
Re: [ccp4bb] alternating strong/weak intensities in reciprocal planes - P622
Hi Jorge, The strong h, k, l=2n and weak h, k, l=2n+1 pattern suggest pseudo body centering. Does the off-origin Patterson peak lie at/near 0.5 0.5 0.5? You could get pseudo body centering if an NCS 2-fold lies parallel to a crystallographic 2(1) or 6(3) screw axis, with the NCS 2-fold a quarter (not half) of a unit cell distant from the crystallographic axis. The fact that you get good merging statistics in P622 even at the high resolution limit suggests to me that you either have that space group or a lower symmetry subgroup with a nearly 0.5 twin fraction. Even if you figure out completely what your pathological crystal conditions are it may be hard to refine the structure properly. In some cases crystals can snap from a pseudo- to a proper crystal by adding the right additive. This may be worth trying while you break your head on this case. One problem is that whenever you make a model that obeys the pseudo body centering you are going to get a significant R-factor and correlation coefficient, even if the actual model is wrong. If you get a clear rotation function solution, which is not affected by the pseudo translation, it may still work but otherwise it could be hard to know if you got the right solution or not. Trying a whole bunch of rotation function solutions and see which one will refine to a significantly lower R-free is one thing to try. Bart Jorge Iulek wrote: Dear all, Please, maybe you could give some suggestions to the problem below. 1) Images show smeared spots, but xds did a good job integrating them. The cell is 229, 229, 72, trigonal, and we see alternating strong and weak rows of spots in the images (spots near each other, but rows more separated, must be by c*). They were scaled with xscale, P622 (no systematic abscences), R_symm = 5.3 (15.1), I/sigI = 34 (14) and redundancy = 7.3 (6.8), resolution 2.8 A. Reciprocal space show strong spots at h, k, l=2n and weak spots at h, k, l=2n+1 (I mean, l=2n intensities are practically all higher than l=2n+1 intensities, as expected from visual inspection of the images). Within planes h, k, l=2n+1, the average intensity is clearly and much *higher at high resolution than at low resolution*. Also, within planes h, k, l=2n, a subjective observation is that average intensity apparently does not decay much from low to high resolution. The data were trucated with truncate, which calculated Wilson B factor to be 35 A**2. 2) Xtriage points a high (66 % of the origin) off-origin Patterson peak. Also, ML estimate of overall B value of F,SIGF = 25.26 A**2. 3) I suspect to have a 2-fold NCS parallel to a (or b), halfway the c parameter, which is almost crystallographic. 4) I submitted the data to the Balbes server which using pseudo-translational symmetry suggested some solutions, one with a good contrast to others, with a 222 tetramer, built from a structure with 40 % identity and 58% positives, of a well conserved fold. 5) I cannot refine below 49 % with either refmac5, phenix.refine or CNS. Maps are messy, except for rather few residues and short stretches near the active site, almost impossible for rebuilding from thereby. Strange, to me, is that all programs freeze all B-factors, taking them the program minimum (CNS lowers to almost its minimum). Might this be due to by what I observed in the reciprocal space as related in 1 ? If so, might my (intensity) scaling procedure have messed the intensities due to their intrinsic property to be stronger in alternating planes ? How to overcome this ? 6) I tried some different scaling strategies *in the refinement step*, no success at all. 7) A Patterson of the solution from Balbes also shows an off-origin Patteron at the same position of the native data, although a little lower. 8) Processed in P6, P312 and P321, all of course suggest twinning. I would thank suggestions, point to similar cases, etc... In fact, currently I wondered why refinement programs take B-factor to such low values Many thanks, Jorge -- == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] alternating strong/weak intensities in reciprocal planes - P622
Hi Jorge, I imagine your 222 tetramer makes a sort of 'pancake' which fits into a cell of 229x229x36 when you apply the 6-fold symmetry. If that was the case in the crystal, then these would be the cell dimensions that you would get. But I suspect you have a situation where the cell repeat has a pancake that is either slightly shifted in the plane normal to the 6-fold, or rotated out of that plane by a small number of degrees. So the crystal would have a doubled unit-cell, with the weak inter-layers. If the two pancakes had been exactly parallel, and exactly 36A apart, the weak layers would have disappeared completely, and the situation would reduce to the smaller cell. Because of the slight translation/rotation between two adjacent smaller cells, you get the weaker layers. The perturbation must be really small that it is much less noticeable at low res, which is where you see the weak reflections in the l=2n+1 layers. As the res goes up, the ability to discern the differences goes up, giving the more intense spots in the outer part of the diffraction pattern. This situation would still occur in the presence of systematic absences due to an unidentified screw axis, as suggested by another contributor. You can try for better ordered crystals, as suggested by someone else. But to rescue this data set, I would look for one good MR solution, then use it as a fixed solution and use the same rotation solution (or one very close to it) to find a second translation solution. This should be within a small fraction of 0,0,0.5. After rigid body refinement, you might see the rotation of the 'pancake' clearly. But to get acceptable R-factors, you must try all the screw axis combinations, 6, 6(1), 6(2), 6(3), 6(4) and 6(5). With a bit of luck, one of these will be much better than the others. One final remark: You seem to have cut off the res at 2.8A despite the significant I/sig(I) statistic in the outer shell, combined with a benign R-merge. This is understandable if it is due to geometry, but really, you must go for higher res, and maybe you will get an even clearer answer. If your data collection system is limiting for the above cell dimension and res combination, you should try a different facility, with a larger detector or shorter wavelength, or both. Synchrotrons are usually good for this sort of thing (of course I am advertising!). Good Luck. Pierre *** Pierre Rizkallah, Daresbury Laboratory, Warrington, Cheshire WA4 4AD, U.K. Phone: (+)44 1925 603808 Fax: (+)44 1925 603124 e-mail: [EMAIL PROTECTED] html: http://www.srs.ac.uk/px/pjr/ -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jorge Iulek Sent: 27 August 2007 12:48 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] alternating strong/weak intensities in reciprocal planes - P622 Dear all, Please, maybe you could give some suggestions to the problem below. 1) Images show smeared spots, but xds did a good job integrating them. The cell is 229, 229, 72, trigonal, and we see alternating strong and weak rows of spots in the images (spots near each other, but rows more separated, must be by c*). They were scaled with xscale, P622 (no systematic abscences), R_symm = 5.3 (15.1), I/sigI = 34 (14) and redundancy = 7.3 (6.8), resolution 2.8 A. Reciprocal space show strong spots at h, k, l=2n and weak spots at h, k, l=2n+1 (I mean, l=2n intensities are practically all higher than l=2n+1 intensities, as expected from visual inspection of the images). Within planes h, k, l=2n+1, the average intensity is clearly and much *higher at high resolution than at low resolution*. Also, within planes h, k, l=2n, a subjective observation is that average intensity apparently does not decay much from low to high resolution. The data were trucated with truncate, which calculated Wilson B factor to be 35 A**2. 2) Xtriage points a high (66 % of the origin) off-origin Patterson peak. Also, ML estimate of overall B value of F,SIGF = 25.26 A**2. 3) I suspect to have a 2-fold NCS parallel to a (or b), halfway the c parameter, which is almost crystallographic. 4) I submitted the data to the Balbes server which using pseudo-translational symmetry suggested some solutions, one with a good contrast to others, with a 222 tetramer, built from a structure with 40 % identity and 58% positives, of a well conserved fold. 5) I cannot refine below 49 % with either refmac5, phenix.refine or CNS. Maps are messy, except for rather few residues and short stretches near the active site, almost impossible for rebuilding from thereby. Strange, to me, is that all programs freeze all B-factors, taking them the program minimum (CNS lowers to almost its minimum). Might this be due to by what I observed in the reciprocal space as related in 1 ? If so, might my (intensity) scaling procedure have messed the intensities due to their intrinsic property
