[ccp4bb] tricky mr problem
Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys
Re: [ccp4bb] tricky mr problem
Taking Tfz of 14.3 I assume that you have used Phaser, and according to what authors say, it is most probably the solution. Maybe map is not yet good, but you can try to refine using modern approaches for refinement of low resolution structures which are recently implemented in Refmac and Phenix. Your map will look better after refinement, as model can move quite significant distance into better position. Phase extension into better resolution data is a method which is developed already the late 80's. So you are in… Good luck FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Sep 24, 2013, at 24:01 , RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys
Re: [ccp4bb] tricky mr problem
On Monday, 23 September, 2013 22:01:32 RHYS GRINTER wrote: Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Apart from whether you need SeMet for phasing, when you are having trouble fitting into poor maps it can help a lot to have the location of methionines pinned down by peaks in an anomalous difference Fourier map. Ethan
Re: [ccp4bb] tricky mr problem
Rhys, Having worked on a number of these structures using both MR and SAD/MAD, my advice is to continue to make experimental phasing a priority since building into a poor density map at this resolution can take months to years to finish while building into a nice experimental map can take a few hours to days, depending on how good your phases are. With that being said, I fully support pursuing the MR route, just keep in mind though it won't easy at this resolution, even if you had awesome data. And yes, your results seem to indicate that you have a nice solution, but i wonder how much of the overall structure does your model cover? You mention this is a 22-stranded beta-barrelmembrane protein? A tonB-dept transporter maybeand if so, do you get a solution with the plug domain or are you just playing with the barrel domain currently? A good check to see if you have a real solution if you are only using the barrel domain is to check to see if you have any difference density for the plug domain. So, my 2 cents, keep chugging along with the MR, but I would put more energy into the experimental phasing, esp if you are able to express it ok and can crystallize using the same conditions as native. Also, both MR and experimental phasing would benefit greatly if you were able to increase your resolution closer to 3 angstroms (ughi know...easier said than done!). Again, if this is indeed a membrane protein, that could just mean that you end up screening a few hundred crystals to find that one crystal with that one sweet spot that gives you that one really good dataset. Good luck with your project, sounds like you are almost there! Cheers, Nick +++ Nicholas Noinaj Laboratory of Molecular Biology NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 Phone (+1) 301-594-9230 Web: http://www-mslmb.niddk.nih.gov/buchanan/index.html From: RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Monday, September 23, 2013 5:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] tricky mr problem Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys
[ccp4bb] Fwd: [ccp4bb] tricky mr problem
I would try phase improvement, especially since you have two molecules per a.u. In other words averaging, solvent flattning, histogram matching. The best way is to start at low(er) resoltuion and extend to the highest resolution vailable. The best criterium for success is a improved and interpretable map. I had quite some success with this appraoch with MR solutions, but uninterpretable or difficult to build maps. Good luck, Klaus Dr. Klaus Piontek Albert-Ludwigs University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de --- the forwarded message follows --- ---BeginMessage--- Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys---End Message---
Re: [ccp4bb] Fwd: [ccp4bb] tricky mr problem
I've had good luck using the CCP4 DM program PARROT to improve poor initial MR maps with twinned data. (Based on some suggestions from this bb.) I think it works especially well if you have a lot of NCS symmetry, but it's worth a try, and doesn't take long. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 9/23/2013 9:01 PM, Klaus Piontek wrote: I would try phase improvement, especially since you have two molecules per a.u. In other words averaging, solvent flattning, histogram matching. The best way is to start at low(er) resoltuion and extend to the highest resolution vailable. The best criterium for success is a improved and interpretable map. I had quite some success with this appraoch with MR solutions, but uninterpretable or difficult to build maps. Good luck, Klaus Dr. Klaus Piontek Albert-Ludwigs University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de --- the forwarded message follows ---
Re: [ccp4bb] tricky mr problem
We often use a S-SAD type data collection (there would be no need for enormous redundancy) which, if your model is correct, should show up the S positions in the same way as a Se-met would show up the Se positions. Good way of validating your model and a help to tracing. Cheers Andy De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ethan A Merritt [merr...@u.washington.edu] Envoyé : lundi 23 septembre 2013 23:25 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] tricky mr problem On Monday, 23 September, 2013 22:01:32 RHYS GRINTER wrote: Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Apart from whether you need SeMet for phasing, when you are having trouble fitting into poor maps it can help a lot to have the location of methionines pinned down by peaks in an anomalous difference Fourier map. Ethan
Re: [ccp4bb] Tricky MR problem
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Rhys, with good 2.0A data you could try S-SAD, or MR-SAD. For the latter I recommend autorickshaw (http://www.embl-hamburg.de/Auto-Rickshaw/). Best, Tim On 10/01/2012 09:26 PM, RHYS GRINTER wrote: Hi All, I'm currently working on solving the structure of a protein by molecular replacement. The protein is around 30kDa and likely has a two beta-prism domains, linked by a long curved two stranded sheet based on the structure of an analogue. There are also a number of other structures which represent a single homologous beta-prism domain. I've tried to find MR solution using the analogue and various truncation/AA substitution models based on it with no success. I've also tried single domain ensembles of the other homologous structures, also with no success. I think the problem is the overall sequence homology is quite low between my protein and the available structures (35% for the analogue and around 20% for the other models. I was curious as to how someone with more experience would tackle this problem. Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQapxiUxlJ7aRr7hoRAnljAKCSi4mxkp0BJ/FvrLQNJszCtLtToACgya1H SN6qckg9//iN2KrNU/oxMXE= =ZOpN -END PGP SIGNATURE-
[ccp4bb] Tricky MR problem
Thanks for your help everyone! It seems that the Balbes pipeline, followed by density modification in Phenix has done the trick Rhys
Re: [ccp4bb] Tricky MR problem
That's good to know, Rhys. But would you mind sharing why did it work with Balbes? Is there a big change in the position of the domains as related to your first searching model, or huge loops that had been removed? Carlos Em 02/10/2012, às 14:42, RHYS GRINTER escreveu: Thanks for your help everyone! It seems that the Balbes pipeline, followed by density modification in Phenix has done the trick Rhys
[ccp4bb] Tricky MR problem
Hi All, I'm currently working on solving the structure of a protein by molecular replacement. The protein is around 30kDa and likely has a two beta-prism domains, linked by a long curved two stranded sheet based on the structure of an analogue. There are also a number of other structures which represent a single homologous beta-prism domain. I've tried to find MR solution using the analogue and various truncation/AA substitution models based on it with no success. I've also tried single domain ensembles of the other homologous structures, also with no success. I think the problem is the overall sequence homology is quite low between my protein and the available structures (35% for the analogue and around 20% for the other models. I was curious as to how someone with more experience would tackle this problem. Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Tricky MR problem
Not sure I understood your problem but it looks like it's related to flexibility. You can try cutting the domains apart (and chopping off the loops) in two different pdbs. They can be used as separate ensembles. Try to find the bigger one first. 35% homology is not that low, just search with polyAla. (You can also analyze the analogue with Normal Modes to see where it bends in case it's not obvious). Carlos Em 01/10/2012, às 21:26, RHYS GRINTER escreveu: Hi All, I'm currently working on solving the structure of a protein by molecular replacement. The protein is around 30kDa and likely has a two beta-prism domains, linked by a long curved two stranded sheet based on the structure of an analogue. There are also a number of other structures which represent a single homologous beta-prism domain. I've tried to find MR solution using the analogue and various truncation/AA substitution models based on it with no success. I've also tried single domain ensembles of the other homologous structures, also with no success. I think the problem is the overall sequence homology is quite low between my protein and the available structures (35% for the analogue and around 20% for the other models. I was curious as to how someone with more experience would tackle this problem. Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Tricky MR problem
I agree with Carlos's suggestions (search for your two domains separately, and as poly-Ala models). There's a few other things that may help MR, which you may or may not have tried: 1. Reset the B-factor of your search models to the wilson B-factor of your dataset. 2. Search using only lower-resolution data (aka if you've got 2.5 - 50, search using 3.5-30 or 4.0-30). 3. If you've got homologous structures for your domains (it sounds like you do), align them and remove any regions that vary significantly from your search model. Model inaccuracy will cause worse problems than model incompleteness, at least in my experience. 4. Search in all alternative spacegroups - it adds a little to the runtime, but IMHO is worth it. Pete RHYS GRINTER wrote: Hi All, I'm currently working on solving the structure of a protein by molecular replacement. The protein is around 30kDa and likely has a two beta-prism domains, linked by a long curved two stranded sheet based on the structure of an analogue. There are also a number of other structures which represent a single homologous beta-prism domain. I've tried to find MR solution using the analogue and various truncation/AA substitution models based on it with no success. I've also tried single domain ensembles of the other homologous structures, also with no success. I think the problem is the overall sequence homology is quite low between my protein and the available structures (35% for the analogue and around 20% for the other models. I was curious as to how someone with more experience would tackle this problem. Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow
Re: [ccp4bb] Tricky MR problem
[1] In addition to using lower resolution data, you should check a variety of RMSDs as well. In fact constructing an RMSD / high resolution contour plot would be beneficial to determine the optimum set of parameters. (That is if you're using PHASER). [2] You say you suspect two molecules per asymmetric unit. Are you fortunate enough to have NCS that's not aligned with the crystal axes? (Have you checked the self rotation patterson?). If there's significant off origin peaks you can (a) try to model the dimer and use that for the search or (b) use the locked rotation function in molrep. [3] Any heavy atoms in the structure (Zn, FeS, etc)? F On Oct 1, 2012, at 12:26 PM, RHYS GRINTER wrote: Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent).
Re: [ccp4bb] Tricky MR problem
Hi Rhys, there is nothing wrong with experimental phases. If a few MR attempts fail, there aren't any real red flags with your crystal form, and you have a few decently diffracting crystals at hand, soak them for a little bit in your favourite heavy atom. We found that soaks in 500-1000mM Na or K iodide in your precipitant solution are very successful. Many crystals tolerate such high iodide concentrations well. Iodide has a very strong anomalous signal in house and binds to a variety of sites. For SSGCID, this has become our main technique to obtain experimental phases. With the great software packages available nowadays, you can have an initial structure within minutes after finishing the data set. For a recent review of this old technique see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836 Best wishes, Jan -- Jan Abendroth Emerald BioStructures / SSGCID Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On Oct 1, 2012, at 12:26 PM, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, I'm currently working on solving the structure of a protein by molecular replacement. The protein is around 30kDa and likely has a two beta-prism domains, linked by a long curved two stranded sheet based on the structure of an analogue. There are also a number of other structures which represent a single homologous beta-prism domain. I've tried to find MR solution using the analogue and various truncation/AA substitution models based on it with no success. I've also tried single domain ensembles of the other homologous structures, also with no success. I think the problem is the overall sequence homology is quite low between my protein and the available structures (35% for the analogue and around 20% for the other models. I was curious as to how someone with more experience would tackle this problem. Just for background, the datasets I have are 2 to 2.7 angstroms with pretty nice stats. The space group is most likely C2221 with two molecules per ASU (giving around 58% solvent). Thanks, Rhys Grinter PhD Candidate University of Glasgow
