Re: [ccp4bb] translational NCS & twinning
Dear all, Thank you very much for all of your suggestions and sharing experiences. As many of you commented, the current small unit cell C2 refinement seems to be incorrect or correct, and I should put some efforts to crack this question. - To Phill Jeffrey, The idea, trying to find high symmetry SG with small unit cell C2 data is good idea, and I will try this. For your last comments, identifiable electron density differences between each chain, I guess there should not be other densities between chains if my current SG and model is correct. Am I right? - To Ethan, Turning off the automatic_tNCS_option seems to be good option. I think, my current data seems to be twinned then tNCS which I am not sure at this moment. But I will keep your advice in my mind. - To Phoebe A. Rice, It is quite interesting that you also could get structure solution by indexing strong spots and having smaller unit cell. Actually, I was wondering how it was possible that having half-sized unit cell could have solution, while full-sized unit cell could not. It will be great if you can share your experience a bit more (e.g the size of smaller unit cell used in initial search for both 1szp and 3pkz) Again, thank you very much for all of your suggestion. Best wishes, Donghyuk To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] multi-xtal averaging
Excellent! And now you can test Xtal averaging with models?? Eleanor On Thu, 10 Jan 2019 at 12:38, Andrew Lovering wrote: > Dear all, > > > > Thanks to Vincent, Eleanor, herman and others! It turns out I’ve managed > to sort it out a different way – the initial TFZ of 10 with cutout density > and resultant map was a bit underwhelming and indicative of an issue – so I > cut my ~140ang long molecule in half and searched with density of those > halves...TFZ increases to 19 and I can now see (and place) the first half > well, with the remainder flexing. I’ll now use these two placed crystal > forms in averaging. A good lesson for me that a tube shaped molecule can > bend between forms > > > > Thanks again > > Andy > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] translational NCS & twinning
For two projects in the distant past, we dealt with tNCS by initially telling lies to the software (1szp and 3pkz). The tNCS was strong enough that there was a clear weak/dark pattern in the diffraction pattern, so for the initial molecular replacement we used a data set in the smaller unit cell where only the strong spots had been boxed and reduced. Then we moved that model to the proper, full unit cell (weak spots included) data set and finished the molecular replacement either by manually pushing the model around according to the appropriate vector(s) or just doing more molrep. If you're desperate, and your weak spots are weak enough, it is worth a try! ~~~ Phoebe A. Rice Dept. of Biochem & Mol. Biol. and Committee on Microbiology https://voices.uchicago.edu/phoebericelab/ On 1/10/19, 12:18 PM, "CCP4 bulletin board on behalf of Ethan A Merritt" wrote: On Thursday, January 10, 2019 2:11:52 AM PST Donghyuk Shin wrote: > Dear all, > > I am having tough time with my Xtal data sets those seem to be twinned or have translational NCS, and it will be greatly appreciated if you can give me some advices or comments! > > Data was initially processed with XDS and scaled with aimless without specifying certain space group (SG). > Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is non-origin peak after patterson analysis. (attached log) > And, I could not get the proper MR-solution from this data sets. > > Because I read that twinning and tNCS cannot be properly distinguished at high SG, I went down to subgroup either P32 or P6 assuming that there is twinning which make data set seems to have apparently high SG. (procedure was same XDS->aimless but I specified the SG to keep them) > Then, xtriage still indicates there is non-origin peak as before, but found twin laws for the data sets (attached log). > However, I still could not get the right MR-solution. > Then, I went even further down to P3 or C2, and xtriage found more twin laws which is make sense because of the lower SG. (attached log) > Again, I could not get the MR-solution. > For all the MR running above, I assumed that phaser(ccp4 module) automatically applied tNCS if they present. or should I have to tick on button in the expert parameters? ^^^ Hi Donghuk, This may indeed be a possible source of problems. I have recent experience with a case where aimless/xtriage/phaser all report a large tNCS component, but allowing phaser to use this information prevents finding a correct MR solution. I can only obtain the known correct solution by telling phaser explicitly _not_ to use tNCS. In my case the true space group is P1 but the angles are all close to 90., causing most indexing programs to falsely identify it as P2. I cannot say how or why the tNCS test triggers, but the known correct solution in P1 does not contain tNCS. Ethan > > Then, I went back to the image and processed the datasets with mosflm by checking the indexed spots. > During this step, I played with the threshold for indexing to follow the strong spot for get correct SG. > I am not sure whether this is correct or not, but by putting high threshold for indexing (e.g. ~15) I could index the data with C2 which has half dimension for a,b axes (116.348, 67.218, 182.861, 90, 90, 90) to the original unit cell (232.533, 134.202, 182.67, 90, 90, 90). > With this, I could put 3 molecules in ASU by MR. During refinement, I felt that the R values were not dropping, and I applied twin refinement. > without twin refinement the R values were (0.39/0.44, work/free), and applying twin refinement gave me significantly better values (0.23/0.26). > Because there were 6 twin operators which may cause this huge R value drop, I speculate whether this is true or not. > > Your comments will be greatly helpful! > > With you all the best, > Donghyuk > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg MS 357742, University of Washington, Seattle 98195-7742 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] translational NCS & twinning
On Thursday, January 10, 2019 2:11:52 AM PST Donghyuk Shin wrote: > Dear all, > > I am having tough time with my Xtal data sets those seem to be twinned or > have translational NCS, and it will be greatly appreciated if you can give me > some advices or comments! > > Data was initially processed with XDS and scaled with aimless without > specifying certain space group (SG). > Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there > is non-origin peak after patterson analysis. (attached log) > And, I could not get the proper MR-solution from this data sets. > > Because I read that twinning and tNCS cannot be properly distinguished at > high SG, I went down to subgroup either P32 or P6 assuming that there is > twinning which make data set seems to have apparently high SG. (procedure was > same XDS->aimless but I specified the SG to keep them) > Then, xtriage still indicates there is non-origin peak as before, but found > twin laws for the data sets (attached log). > However, I still could not get the right MR-solution. > Then, I went even further down to P3 or C2, and xtriage found more twin laws > which is make sense because of the lower SG. (attached log) > Again, I could not get the MR-solution. > For all the MR running above, I assumed that phaser(ccp4 module) > automatically applied tNCS if they present. or should I have to tick on > button in the expert parameters? ^^^ Hi Donghuk, This may indeed be a possible source of problems. I have recent experience with a case where aimless/xtriage/phaser all report a large tNCS component, but allowing phaser to use this information prevents finding a correct MR solution. I can only obtain the known correct solution by telling phaser explicitly _not_ to use tNCS. In my case the true space group is P1 but the angles are all close to 90., causing most indexing programs to falsely identify it as P2. I cannot say how or why the tNCS test triggers, but the known correct solution in P1 does not contain tNCS. Ethan > > Then, I went back to the image and processed the datasets with mosflm by > checking the indexed spots. > During this step, I played with the threshold for indexing to follow the > strong spot for get correct SG. > I am not sure whether this is correct or not, but by putting high threshold > for indexing (e.g. ~15) I could index the data with C2 which has half > dimension for a,b axes (116.348, 67.218, 182.861, 90, 90, 90) to the > original unit cell (232.533, 134.202, 182.67, 90, 90, 90). > With this, I could put 3 molecules in ASU by MR. During refinement, I felt > that the R values were not dropping, and I applied twin refinement. > without twin refinement the R values were (0.39/0.44, work/free), and > applying twin refinement gave me significantly better values (0.23/0.26). > Because there were 6 twin operators which may cause this huge R value drop, I > speculate whether this is true or not. > > Your comments will be greatly helpful! > > With you all the best, > Donghyuk > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg MS 357742, University of Washington, Seattle 98195-7742 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] translational NCS & twinning
Donghyuk The combination of two things gives me cause for concern: 1. You've reindexed something that apparently scaled OK in point group 622 into point group 2, with a smaller cell. Since it's hard to fake that sort of data agreement in 622, I assume your data is at the very least pseudo-622. 2. You've modeled that additional symmetry using a whole array of twin operators and some non-crystallographic symmetry. This may in fact be the correct model, but there's a significant risk that you're inappropriately modeling something. Let's assume for a moment that your small-cell C2 refinement with 6 twin ops improves the model somewhat. Now go back and generate scaled data sets in all possible point groups suggested by Pointless for that data, and try and find molecular replacement solutions with your partially-refined model by testing every possible space group in every possible point group based on the Pointless suggestions. The idea is to try and model more of the 622 pseudo-symmetry as crystallographic symmetry, using fewer twin operators in refinement. If you test all of these combinations, which might be quite extensive, you might find one or more that fit nearly as well as your current C2 solution and has higher symmetry. You should take a hard look at that those potential solutions. Only when you've thoroughly exhausted alternative molecular replacement solutions would you be confident that your C2 model is in fact the only reasonable explanation of your data. But as it stands it is rather atypical and it warrants further investigation Additional evidence that your C2 cell is the only reasonable model would be identifiable electron density differences between each chain in your (presumed) multi-chain model. Cheers Phil Jeffrey Princeton On 1/10/19 11:08 AM, Donghyuk Shin wrote: Dear Jacob Keller and Vipul, Thank you both very much for the reply. Regarding the R-values, I am just wondering whether the huge gap between refinements w/ w/o twin operator can be possible even the crystal is not twinned? Best wishes, Donghyuk To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] AW: [EXTERNAL] [ccp4bb] translational NCS & twinning
Dear Donghyuk, Unfortunately, everything is possible when NCS, twinning etc. get into the game. I do not have answers, but some questions for you to think about: - Do you really have 6 twinning operators, or only one operator and are the other operators generated by (non)crystallographic symmetry? - Both your P6322 cell and your C2 cell have angles of 90 90 90. For P6322, the last angle should be 120 and for C2, only the second angle is constrained to be 90. Maybe you should check that not somewhere something went wrong with the cell angles. - How weak are the reflections that got discarded by halving the a- and b-axes? Do they have significant intensity, or is it only noise? - By shrinking the unit cell, you may have created artificial twinning when in the large unit cell the molecules are related by "twinning" (N)CS. - Since you seem to have found a solution with the small unit cell, you could see if you could fit this solution in the large unit cell: Process in P1 in the large unit cell and use the ensemble (the complete! unit cell of your C2 solution, as a search model. - Your current solution maybe correct after all, but I would analyze it very critically. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Donghyuk Shin Gesendet: Donnerstag, 10. Januar 2019 11:12 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] translational NCS & twinning Dear all, I am having tough time with my Xtal data sets those seem to be twinned or have translational NCS, and it will be greatly appreciated if you can give me some advices or comments! Data was initially processed with XDS and scaled with aimless without specifying certain space group (SG). Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is non-origin peak after patterson analysis. (attached log) And, I could not get the proper MR-solution from this data sets. Because I read that twinning and tNCS cannot be properly distinguished at high SG, I went down to subgroup either P32 or P6 assuming that there is twinning which make data set seems to have apparently high SG. (procedure was same XDS->aimless but I specified the SG to keep them) Then, xtriage still indicates there is non-origin peak as before, but found twin laws for the data sets (attached log). However, I still could not get the right MR-solution. Then, I went even further down to P3 or C2, and xtriage found more twin laws which is make sense because of the lower SG. (attached log) Again, I could not get the MR-solution. For all the MR running above, I assumed that phaser(ccp4 module) automatically applied tNCS if they present. or should I have to tick on button in the expert parameters? Then, I went back to the image and processed the datasets with mosflm by checking the indexed spots. During this step, I played with the threshold for indexing to follow the strong spot for get correct SG. I am not sure whether this is correct or not, but by putting high threshold for indexing (e.g. ~15) I could index the data with C2 which has half dimension for a,b axes (116.348, 67.218, 182.861, 90, 90, 90) to the original unit cell (232.533, 134.202, 182.67, 90, 90, 90). With this, I could put 3 molecules in ASU by MR. During refinement, I felt that the R values were not dropping, and I applied twin refinement. without twin refinement the R values were (0.39/0.44, work/free), and applying twin refinement gave me significantly better values (0.23/0.26). Because there were 6 twin operators which may cause this huge R value drop, I speculate whether this is true or not. Your comments will be greatly helpful! With you all the best, Donghyuk To unsubscribe from the CCP4BB list, click the following link: https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwIFaQ=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=xlZKtKm2eNS0qmQb4wXnIIknGL1-n0M0toJoWb2Ezco=scEheHieLBLhJMaXoEDimlYM9TAk600XwBjYXrQC1nc= To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] translational NCS & twinning
Dear Jacob Keller and Vipul, Thank you both very much for the reply. Regarding the R-values, I am just wondering whether the huge gap between refinements w/ w/o twin operator can be possible even the crystal is not twinned? Best wishes, Donghyuk To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] PhD position in Structural and Chemical Biology at St Jude
The Fischer lab at St. Jude Children’s Research Hospital is recruiting a graduate student for a project that exploits protein dynamics for ligand discovery against pediatric cancer targets. You will be part of a young interdisciplinary team that uses structural biology (crystallography, NMR), protein biochemistry, and single-molecule methods (smFRET, HDX-MS) to characterize dynamic, disease-relevant protein states. By finding ligands against those flexible protein states, and revealing allosteric networks we can explore new ways to modulate protein malfunction in disease. The project will be housed in the Department of Chemical Biology and Therapeutics ( https://www.stjude.org/research/departments-divisions/chemical-biology-therapeutics.html), and Structural Biology ( https://www.stjude.org/research/departments-divisions/structural-biology.html) at St. Jude Children’s Research Hospital. With ongoing investments into exceptional structural biology, chemical biology and computational facilities, this is an exciting time to join the lab. Accompanying courses will be taken in conjunction with the Department of Pharmaceutical Sciences at the University of Tennessee Health Science Center. This work will provide a solid introduction to current opportunities in structure-based ligand discovery with relevance for both academia and industry. With its excellent core facilities, highly interactive and supportive environment St. Jude Children’s Research Hospital is a great place to do research and build a career whilst living in an affordable city. Admission requirements: - a BS or MS degree in pharmacy, chemistry, biology, mathematics, engineering, or other appropriate disciplines. - a minimum Grade Point Average of 3.0 - a combined Graduate Record Examination score (verbal and quantitative) of at least 300 - proof of proficiency in English (e.g. TOEFL) Please direct your questions and application package including a cover letter, current CV, and 3 letters of reference to: Dr. Marcus Fischer ( marcus.fisc...@stjude.org) before the end of February 2019. Online application deadline is March 15thfor admission to the Fall Semester starting in August 2019. Relevant papers include: • Fischer et al. (2014). Incorporation of protein flexibility & conformational energy penalties in docking screens to improve ligand discovery. *Nature Chemistry*6, 575-83. PMID 24950326 • Balius et al. (2017). Testing inhomogeneous solvation theory in structure-based ligand discovery. *PNAS*E6839-46. PMID 28760952 • Fischer et al. (2015). One crystal, two temperatures: cryocooling penalties alter ligand binding to transient protein sites. *Chembiochem*1560-64. PMID 26032594 More info at: https://www.stjude.org/fischer To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] translational NCS & twinning
>>I feel you went ahead with right strategy. I agree with this part regarding lowering symmetry. >>For 2.1 A datasrt, the appropriate drop in Rfree/ Rwork is a strong >>indicator, i believe. This is not true—even non-twinned data will improve in R values with twinning operators added as parameters. And twin-refined structures always have lower R values. JPK On Thu 10 Jan, 2019, 3:52 PM Donghyuk Shin mailto:sdh...@gmail.com> wrote: Dear all, I am having tough time with my Xtal data sets those seem to be twinned or have translational NCS, and it will be greatly appreciated if you can give me some advices or comments! Data was initially processed with XDS and scaled with aimless without specifying certain space group (SG). Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates there is non-origin peak after patterson analysis. (attached log) And, I could not get the proper MR-solution from this data sets. Because I read that twinning and tNCS cannot be properly distinguished at high SG, I went down to subgroup either P32 or P6 assuming that there is twinning which make data set seems to have apparently high SG. (procedure was same XDS->aimless but I specified the SG to keep them) Then, xtriage still indicates there is non-origin peak as before, but found twin laws for the data sets (attached log). However, I still could not get the right MR-solution. Then, I went even further down to P3 or C2, and xtriage found more twin laws which is make sense because of the lower SG. (attached log) Again, I could not get the MR-solution. For all the MR running above, I assumed that phaser(ccp4 module) automatically applied tNCS if they present. or should I have to tick on button in the expert parameters? Then, I went back to the image and processed the datasets with mosflm by checking the indexed spots. During this step, I played with the threshold for indexing to follow the strong spot for get correct SG. I am not sure whether this is correct or not, but by putting high threshold for indexing (e.g. ~15) I could index the data with C2 which has half dimension for a,b axes (116.348, 67.218, 182.861, 90, 90, 90) to the original unit cell (232.533, 134.202, 182.67, 90, 90, 90). With this, I could put 3 molecules in ASU by MR. During refinement, I felt that the R values were not dropping, and I applied twin refinement. without twin refinement the R values were (0.39/0.44, work/free), and applying twin refinement gave me significantly better values (0.23/0.26). Because there were 6 twin operators which may cause this huge R value drop, I speculate whether this is true or not. Your comments will be greatly helpful! With you all the best, Donghyuk To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] multi-xtal averaging
Dear all, Thanks to Vincent, Eleanor, herman and others! It turns out I've managed to sort it out a different way - the initial TFZ of 10 with cutout density and resultant map was a bit underwhelming and indicative of an issue - so I cut my ~140ang long molecule in half and searched with density of those halves...TFZ increases to 19 and I can now see (and place) the first half well, with the remainder flexing. I'll now use these two placed crystal forms in averaging. A good lesson for me that a tube shaped molecule can bend between forms Thanks again Andy To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] translational NCS & twinning
Hi Donghyuk, I feel you went ahead with right strategy. For 2.1 A datasrt, the appropriate drop in Rfree/ Rwork is a strong indicator, i believe. If you have already build all possible model, using tls can be of further help. Cheers, Vipul On Thu 10 Jan, 2019, 3:52 PM Donghyuk Shin Dear all, > > I am having tough time with my Xtal data sets those seem to be twinned or > have translational NCS, and it will be greatly appreciated if you can give > me some advices or comments! > > Data was initially processed with XDS and scaled with aimless without > specifying certain space group (SG). > Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates > there is non-origin peak after patterson analysis. (attached log) > And, I could not get the proper MR-solution from this data sets. > > Because I read that twinning and tNCS cannot be properly distinguished at > high SG, I went down to subgroup either P32 or P6 assuming that there is > twinning which make data set seems to have apparently high SG. (procedure > was same XDS->aimless but I specified the SG to keep them) > Then, xtriage still indicates there is non-origin peak as before, but > found twin laws for the data sets (attached log). > However, I still could not get the right MR-solution. > Then, I went even further down to P3 or C2, and xtriage found more twin > laws which is make sense because of the lower SG. (attached log) > Again, I could not get the MR-solution. > For all the MR running above, I assumed that phaser(ccp4 module) > automatically applied tNCS if they present. or should I have to tick on > button in the expert parameters? > > Then, I went back to the image and processed the datasets with mosflm by > checking the indexed spots. > During this step, I played with the threshold for indexing to follow the > strong spot for get correct SG. > I am not sure whether this is correct or not, but by putting high > threshold for indexing (e.g. ~15) I could index the data with C2 which has > half dimension for a,b axes (116.348, 67.218, 182.861, 90, 90, 90) to the > original unit cell (232.533, 134.202, 182.67, 90, 90, 90). > With this, I could put 3 molecules in ASU by MR. During refinement, I felt > that the R values were not dropping, and I applied twin refinement. > without twin refinement the R values were (0.39/0.44, work/free), and > applying twin refinement gave me significantly better values (0.23/0.26). > Because there were 6 twin operators which may cause this huge R value > drop, I speculate whether this is true or not. > > Your comments will be greatly helpful! > > With you all the best, > Donghyuk > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] FW: complex multi-crystal averaging
Dear Andy, for the I222 crystal, you mention a large difference in diffracting resolution, but staraniso doesn't help. Maybe you simply don't have anisotropy, but rather lack of completeness in high resolution shells, a different problem. How much is the anisotropy value given by Phaser? I got confused by your description of what you have and don't have. Maybe the simplest would be to build as good as you can in your P3212, and use this as a MR search for the I222 crystal. Phaser will take care of things for you. Your anisotropy is strong but others have managed with these values so keep faith, you can still get something out of it. Start building in Calpha, and iterative cycles of manual building and refinement will get you to a good place. If you have alpha helices, it is much easier than beta sheets, so focus on those. I wouldn't give up on a 3A dataset, even with large difference in diffracting resolutions. Good luck. Vincent On 10/01/2019 12:05, Andrew Lovering wrote: Ok – small oversight – I can see now that phenix has the atoms placed/saved during cutout stage – so how best to use PHASER output to shift this file? Andy *From:*Andrew Lovering (School of Biosciences) *Sent:* 10 January 2019 10:48 *To:* 'ccp4bb@jiscmail.ac.uk' *Subject:* RE: complex multi-crystal averaging Thanks everyone for the pointers. I should clarify – my issue here is the multiple stage “disconnect” between PDB in xtal1 -> cutout density (into a “interim cell” just for this stage) -> phaser MR with density search model into xtal2 Hence, do I just simply pop the model from (1) into the “interim cell” (display cutout density, move molecule here in coot, bit of manual shifting / rigid body, save interim PDB) then use PDBSET on saved PDB with angles and shifts from phaser output? To use clear language, “what’s the easiest way to get PDB xtal1 to match placed density of MR in xtal2” J Andy *From:*Andrew Lovering (School of Biosciences) *Sent:* 09 January 2019 16:08 *To:* 'ccp4bb@jiscmail.ac.uk' *Subject:* complex multi-crystal averaging Dear All, I suspect the way out of this is a new crystal (!) but interested to hear any advice. I have two crystal forms of a 500aa protein, vaguely tube-shaped 1=P3121, diffracts to 4.1 ang, 3 copies in asu, 86% solvent; map indicates it is a relative of other folds (but those are not so close that they’d be a good guide to sequence register). I have selenomet SAD phases which helps identify Met positions. The 3-fold and high solvent give a great map but you wouldn’t want to build it and buccanner and phenix think similar 2=I222, 2 copies per asu, 66% solvent. This has a cell that gives wildly anisotropic diffraction – ~3, 3.5, 4.3 down different axes. Not really rectified by staraniso. No phases So I can cut the density out of form 1 map (using secondary structure elements of a rough PDB as a mask), and use phaser with this density as search model to find the two copies with a TFZ of about 10. The phaser map shows a bit of detail and the solution has placed the protomers such that they agree with a self-rotation function. Phenix find_ncs on phaser map similarly agrees (as I would expect). Now...I have an eye on multi-crystal averaging between the two forms. BUT the phaser map isn’t good enough to manually place the PDB used in cut-out density, and I can’t see a straightforward way of using the angle info in phaser sol to perform a co-ordinate transformation (but I think ccp4bb users will put me right on this – I’d imagine placing the PDB into the cutout density mtz then using PDBset?). I tried converting the phaser mtz to a map using FFT then using phased TF in Molrep to place the PDB but this didn’t work, complaining about the grid used. One last caveat – I have multiple sets for the I222 that intriguingly differ by 12 angstrom down a 240 ang axis: doing multi-crystal averaging between these two forms achieves little when I would expect otherwise (all the NCS correlations are good, high initial agreement) Like I said...fingers crossed for a new crystal form! Andy To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- Vincent Chaptal, PhD MMSB -UMR5086 Drug Resistance and Membrane Proteins Laboratory 7 passage du Vercors 69007 LYON FRANCE +33 4 37 65 29 01 http://mmsb.cnrs.fr/en/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] FW: complex multi-crystal averaging
Ok - small oversight - I can see now that phenix has the atoms placed/saved during cutout stage - so how best to use PHASER output to shift this file? Andy From: Andrew Lovering (School of Biosciences) Sent: 10 January 2019 10:48 To: 'ccp4bb@jiscmail.ac.uk' Subject: RE: complex multi-crystal averaging Thanks everyone for the pointers. I should clarify - my issue here is the multiple stage "disconnect" between PDB in xtal1 -> cutout density (into a "interim cell" just for this stage) -> phaser MR with density search model into xtal2 Hence, do I just simply pop the model from (1) into the "interim cell" (display cutout density, move molecule here in coot, bit of manual shifting / rigid body, save interim PDB) then use PDBSET on saved PDB with angles and shifts from phaser output? To use clear language, "what's the easiest way to get PDB xtal1 to match placed density of MR in xtal2" :) Andy From: Andrew Lovering (School of Biosciences) Sent: 09 January 2019 16:08 To: 'ccp4bb@jiscmail.ac.uk' Subject: complex multi-crystal averaging Dear All, I suspect the way out of this is a new crystal (!) but interested to hear any advice. I have two crystal forms of a 500aa protein, vaguely tube-shaped 1=P3121, diffracts to 4.1 ang, 3 copies in asu, 86% solvent; map indicates it is a relative of other folds (but those are not so close that they'd be a good guide to sequence register). I have selenomet SAD phases which helps identify Met positions. The 3-fold and high solvent give a great map but you wouldn't want to build it and buccanner and phenix think similar 2=I222, 2 copies per asu, 66% solvent. This has a cell that gives wildly anisotropic diffraction - ~3, 3.5, 4.3 down different axes. Not really rectified by staraniso. No phases So I can cut the density out of form 1 map (using secondary structure elements of a rough PDB as a mask), and use phaser with this density as search model to find the two copies with a TFZ of about 10. The phaser map shows a bit of detail and the solution has placed the protomers such that they agree with a self-rotation function. Phenix find_ncs on phaser map similarly agrees (as I would expect). Now...I have an eye on multi-crystal averaging between the two forms. BUT the phaser map isn't good enough to manually place the PDB used in cut-out density, and I can't see a straightforward way of using the angle info in phaser sol to perform a co-ordinate transformation (but I think ccp4bb users will put me right on this - I'd imagine placing the PDB into the cutout density mtz then using PDBset?). I tried converting the phaser mtz to a map using FFT then using phased TF in Molrep to place the PDB but this didn't work, complaining about the grid used. One last caveat - I have multiple sets for the I222 that intriguingly differ by 12 angstrom down a 240 ang axis: doing multi-crystal averaging between these two forms achieves little when I would expect otherwise (all the NCS correlations are good, high initial agreement) Like I said...fingers crossed for a new crystal form! Andy To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] complex multi-crystal averaging
Thanks everyone for the pointers. I should clarify - my issue here is the multiple stage "disconnect" between PDB in xtal1 -> cutout density (into a "interim cell" just for this stage) -> phaser MR with density search model into xtal2 Hence, do I just simply pop the model from (1) into the "interim cell" (display cutout density, move molecule here in coot, bit of manual shifting / rigid body, save interim PDB) then use PDBSET on saved PDB with angles and shifts from phaser output? To use clear language, "what's the easiest way to get PDB xtal1 to match placed density of MR in xtal2" :) Andy From: Andrew Lovering (School of Biosciences) Sent: 09 January 2019 16:08 To: 'ccp4bb@jiscmail.ac.uk' Subject: complex multi-crystal averaging Dear All, I suspect the way out of this is a new crystal (!) but interested to hear any advice. I have two crystal forms of a 500aa protein, vaguely tube-shaped 1=P3121, diffracts to 4.1 ang, 3 copies in asu, 86% solvent; map indicates it is a relative of other folds (but those are not so close that they'd be a good guide to sequence register). I have selenomet SAD phases which helps identify Met positions. The 3-fold and high solvent give a great map but you wouldn't want to build it and buccanner and phenix think similar 2=I222, 2 copies per asu, 66% solvent. This has a cell that gives wildly anisotropic diffraction - ~3, 3.5, 4.3 down different axes. Not really rectified by staraniso. No phases So I can cut the density out of form 1 map (using secondary structure elements of a rough PDB as a mask), and use phaser with this density as search model to find the two copies with a TFZ of about 10. The phaser map shows a bit of detail and the solution has placed the protomers such that they agree with a self-rotation function. Phenix find_ncs on phaser map similarly agrees (as I would expect). Now...I have an eye on multi-crystal averaging between the two forms. BUT the phaser map isn't good enough to manually place the PDB used in cut-out density, and I can't see a straightforward way of using the angle info in phaser sol to perform a co-ordinate transformation (but I think ccp4bb users will put me right on this - I'd imagine placing the PDB into the cutout density mtz then using PDBset?). I tried converting the phaser mtz to a map using FFT then using phased TF in Molrep to place the PDB but this didn't work, complaining about the grid used. One last caveat - I have multiple sets for the I222 that intriguingly differ by 12 angstrom down a 240 ang axis: doing multi-crystal averaging between these two forms achieves little when I would expect otherwise (all the NCS correlations are good, high initial agreement) Like I said...fingers crossed for a new crystal form! Andy To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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