[ccp4bb] co-crystallization
Hi , I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)? Any suggestions would help. Thanks TY
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Re: [ccp4bb] co-crystallization
for me, I prefer to sock these compounds into your crystal. it will much more easy than co-crystallizaiton. But each protein should be different. Normally when I star to co-crystallization with small compound, I will set up the complex with 1:1.2 molar ratio as first trial to see what should happen. As DMESO, you can't get rid of them. But you can find as much lower concentraiton as you can.
Re: [ccp4bb] number of cycles in refmac
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Ian, I dare say that the goal is to get phases which match as good as possible with what is inside the crystal. If this coincides with maximising the likelihood, why don't we run refinement until the LL stabilises? @Garib: I have seen runs where Refmac actually does stop prematurely, i.e. before the number of cycles set in the input script. It seems that it stops when LL does not drop between two cycles, but according to Ian this would be the point to reach. My question: does this indeed provide the best interpretable map (be it cheating or not)? Cheers, Tim On 08/24/2011 05:36 PM, Ian Tickle wrote: Hi Tim The answer is a definite NO, the goal of refinement is to maximise the likelihood from the working set and the restraints. It is definitely not to optimise Rfree or LLfree. The correct value of the latter is whatever you get at convergence of refinement, i.e. at maximum of the likelihood, anything else is 'cheating'. Cheers -- Ian On Wed, Aug 24, 2011 at 4:24 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear all, especially at the beginning of model building and/or at low resolution both Rfree and -LL free as reported in the refmac logfile show a minimum at a some cycle before rising again. I am certainly not the only one tempted to first run refmac with a large number of refinement cycles, determine that minimum and rerun refmac with ncyc set to that minimum. Of course I want the resulting model and phases/map to be as close to the what's in the crystal as possible in order to facilitate model building. Is it therefore good practice to interrupt refmac wherever it finds a minimum (if so, the minimum w.r.t. which number reported in the log-file)? Thanks for everyone's opinion and experience, Tim - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOVgiBUxlJ7aRr7hoRAq+dAKDj2B6iUMD7C4uu8UMznTlKoclYzACdF8nP Q6DmIFGPcfoP6xbJRwooWWI= =7r5T -END PGP SIGNATURE-
Re: [ccp4bb] Creating CIF Files and Renaming Ligands
Did you rename the ligand in the CIF file as well? It would be nice if the Prodrg interface allowed you to type in the name of the ligand you want in the CIF and PDB file. Hopefully a global replace in your favourite text editor of DRG with LIG or whatever you want to call your ligand would work. You would have to do that on the separate pdb and CIF files and then merge the two or more CIF files into one if you have more than one ligand. I must admit to this being theory I have not tested it in practice. Best wishes Nick -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
[ccp4bb] Melting point and heat of fusion
Dear, We're wondering what tools there are available in order to estimate the 'melting point' and 'heat of fusion' from a known crystal structure (e.g. coordinate file)..? It concerns mostly small molecules ( 100 atoms). Many thanks Kristof --- Kristof Van Hecke, PhD Biomolecular Architecture Celestijnenlaan 200F 3001 Heverlee (Leuven) Belgium ---
Re: [ccp4bb] number of cycles in refmac
Hello Tim It was in one or two versions and I did not get consistent results. However code is there and I can activate it if you want. If you know what criteria you would like to use I can code that also. In some cases it happens that R/Rfree go up and then they start coming down. It may be case when starting model has not very nice geometry. We are minimising total function. And there are several hidden parameters in the total function (weights between Xray and geometry etc), they may cause problems. Cheers Garib On 25 Aug 2011, at 10:32, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Ian, I dare say that the goal is to get phases which match as good as possible with what is inside the crystal. If this coincides with maximising the likelihood, why don't we run refinement until the LL stabilises? @Garib: I have seen runs where Refmac actually does stop prematurely, i.e. before the number of cycles set in the input script. It seems that it stops when LL does not drop between two cycles, but according to Ian this would be the point to reach. My question: does this indeed provide the best interpretable map (be it cheating or not)? Cheers, Tim On 08/24/2011 05:36 PM, Ian Tickle wrote: Hi Tim The answer is a definite NO, the goal of refinement is to maximise the likelihood from the working set and the restraints. It is definitely not to optimise Rfree or LLfree. The correct value of the latter is whatever you get at convergence of refinement, i.e. at maximum of the likelihood, anything else is 'cheating'. Cheers -- Ian On Wed, Aug 24, 2011 at 4:24 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear all, especially at the beginning of model building and/or at low resolution both Rfree and -LL free as reported in the refmac logfile show a minimum at a some cycle before rising again. I am certainly not the only one tempted to first run refmac with a large number of refinement cycles, determine that minimum and rerun refmac with ncyc set to that minimum. Of course I want the resulting model and phases/map to be as close to the what's in the crystal as possible in order to facilitate model building. Is it therefore good practice to interrupt refmac wherever it finds a minimum (if so, the minimum w.r.t. which number reported in the log-file)? Thanks for everyone's opinion and experience, Tim - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.11 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOVgiBUxlJ7aRr7hoRAq+dAKDj2B6iUMD7C4uu8UMznTlKoclYzACdF8nP Q6DmIFGPcfoP6xbJRwooWWI= =7r5T -END PGP SIGNATURE- Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Creating CIF Files and Renaming Ligands
Hi all, Thanks for the suggestions! Anyway, the problems have been fixed! When I edited the CIF files in a mac editor, the file formatting got changed, so a line feed became a carriage return and Coot does not like this as to it they appear to be different files. The CIF files were fixed by using gedit on the old files on linux machine and global replace with match case/whole word only option on. They now are recognized by COOT. Ai Fen On Thu, Aug 25, 2011 at 8:45 PM, Nicholas Keep n.k...@mail.cryst.bbk.ac.ukwrote: Did you rename the ligand in the CIF file as well? It would be nice if the Prodrg interface allowed you to type in the name of the ligand you want in the CIF and PDB file. Hopefully a global replace in your favourite text editor of DRG with LIG or whatever you want to call your ligand would work. You would have to do that on the separate pdb and CIF files and then merge the two or more CIF files into one if you have more than one ligand. I must admit to this being theory I have not tested it in practice. Best wishes Nick -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
Re: [ccp4bb] number of cycles in refmac
TIm, I dare say that the goal is to get phases which match as good as possible with what is inside the crystal. If this coincides with maximising the likelihood, why don't we run refinement until the LL stabilises? That's exactly what you should do: any optimisation procedure attains the correct solution only when changes in the parameters become 'insignificant' i.e. the optimisation is deemed to have converged. This corresponds to the extremum in the target function being attained so that the gradients w.r.t. the refined parameters become zero (within some margin of numerical precision), A very simple 1-parameter analogy is Hero's method to obtain the square root of a number (named after the 1st century Greek mathematician Hero of Alexandria though attributed to the Babylonians: http://en.wikipedia.org/wiki/Babylonian_method). This uses 'fixed-point iteration' to optimise the initial estimate X of sqrt(A) (X can be any positive number, the method has infinite radius of convergence): just replace X by the new estimate (X+A/X)/2 and iterate to convergence. So let's say I want sqrt(2): I make an initial guess of 1, so (1+2/1)/2 = 1.5, then (1.5+2/1.5)/2 = 1.4167 and keep iterating until the result doesn't change: only then will you have the correct answer. Since the target function in MX refinement is the total likelihood (working set + restraints), there's no reason whatsoever why any another function, such as Rfree LLfree, should have an extremum at the same point in parameter space as the target function. Rfree is particular is problematic because it is unweighted, so poorly measured reflections in the test set are going to have a disproportionate influence on the result (e.g. see *Acta Cryst.* (1970). A*26*, 162). Cheers -- Ian
Re: [ccp4bb] number of cycles in refmac
Hmm, I used to think I understood this, but I'm feeling a bit dim right now. On 25/08/2011 11:07, Ian Tickle wrote: Since the target function in MX refinement is the total likelihood (working set + restraints), there's no reason whatsoever why any another function, such as Rfree LLfree, should have an extremum at the same point in parameter space as the target function. This is self-evident; what is not obvious is why the target function should be having the final word. Wasn't the word over-refinement introduced to describe exactly this: that the target function was wrong? Isn't this the purpose of cross-validation, to use an independent measure to judge when the refinement is /not/ producing the best model? Rfree is particular is problematic because it is unweighted, so poorly measured reflections in the test set are going to have a disproportionate influence on the result (e.g. see /Acta Cryst./ (1970). A*26*, 162). This may be true; but as it is independent of refinement, is it not nevertheless the only measure I should trust? Or maybe what you intended to say: only trust refinements for which Rfree decreases monotonically, because only then do you have a valid choice of parameters. phx.
Re: [ccp4bb] number of cycles in refmac
Hi Frank This is self-evident; what is not obvious is why the target function should be having the final word. Wasn't the word over-refinement introduced to describe exactly this: that the target function was wrong? I assumed people were confusing 'over-refinement' with 'over-fitting'; there is no such thing as over-refinement (you won't find 'over-optimisation' mentioned in any textbooks on optimisation!), since by definition refining beyond convergence is a waste of time: it cannot result in any further significant changes in the parameters. Refinement is simply a method of solving a set of equations clearly we want to solve those equations as accurately as possible. If we could obtain an exact solution without iteration we would use that; the mechanics of refinement and the path that the program by dint of numerical quirks happens to take to reach the solution are irrelevant as far as the user is concerned. Stopping the refinement before convergence will not produce more accurate parameters, it will just introduce random and unquantifiable errors. Isn't this the purpose of cross-validation, to use an independent measure to judge when the refinement is *not* producing the best model? If the value of your chosen X-validation metric at convergence indicates a problem with the model, parameterisation, weighting etc then clearly the target function is not indeed the final word: the solution is to fix whatever is wrong and do the refinement again, until you get a satisfactory value for your metric. This may be true; but as it is independent of refinement, is it not nevertheless the only measure I should trust? No there several possible functions of the test set (e.g. Hamilton Rfree, LLfree) that you could use, all potentially equally valid X-validation metrics. I would have more faith in a function such as LLfree in which the contributions of the reflections are at least weighted according to their reliability. It just seems bizarre that important decisions are being based on measurements that may have gross errors without taking those errors into account. Or maybe what you intended to say: only trust refinements for which Rfree decreases monotonically, because only then do you have a valid choice of parameters. No, as I indicated above, what Rfree does before convergence is attained is totally meaningless, only the value obtained _at_ convergence is meaningful as a X-validation statistic. We wouldn't be having this discussion if the refinement program omitted the meaningless intermediate values and only printed out the final Rfree or LLfree. I'm saying that Rfree is not the best X-validation metric because poorly measured data are not properly weighted: this is what Acta paper I referenced is saying. Cheers -- Ian
[ccp4bb] loop building with ARP/wARP
Hi all, I have a large disordered loop (33aa) for a 2.0 Å dataset for which the rest of the structure is well-defined, and phases are decent (Rwork=19.6, Rfree=24.6). I can see some broken up density at one end, but have been unable to convincingly build into into this region manually. I would like to try arp/warp to improve density or possibly extend the loop termini but have been thwarted by some technical problems and would appreciate any advice. I've installed ccp4 6.2.0 on Mac OS X (10.6.8) with fink, both as 32-bit and 64-bit to allow me to run ARP/wARP (version 7.1) from the ccp4i GUI. I've been unable to get loopy to work, and also the standard ARP/wARP classic. When I run ARP/wARP Loops (a.k.a. loopy), I get the message in the log file: Couldn't find any loop to build, loopy will simple copy the pdb file. The program is able to read the sequence .pir file and match it to the structure, and seems from the sequence alignment to identify the regions of the sequence for which there is no structure. I'm guessing that I'm not putting some obvious parameter into the GUI/starting script, but I don't know what that might be. The script generated from the GUI (21_loopy.def) is pasted below. When I try to run ARP/wARP classic for loop building, I get the following message in the logfile: QUITTING ... ARP/wARP module stopped with an error message: REFMAC *** Look for error message in the file: /Users/gbowman/Greg/20_warpNtrace_refine.last.log At the end of the warpNtrace_refine.last.log file, the program reports what seem to be close contacts. Since I don't see this in the structure, and don't have any problems or errors like this when I refine the structure with REMAC, is this from rebuilding, and is this the problem that halts REFMAC? Thanks! Greg = = = = = = 20_warpNtrace_refine.last.log = = = = = = snip Input file :restraints.pdb NUMBER OF MONOMERS IN THE LIBRARY : 11465 with complete description: 11465 NUMBER OF MODIFICATIONS:53 NUMBER OF LINKS:66 I am reading libraries. Please wait. - energy parameters - monomers description (links mod ) BFONT COLOR=#FF!--SUMMARY_BEGIN-- !--SUMMARY_END--/FONT/B Number of atoms:2463 Number of residues : 859 Number of chains : 3 I am reading library. Please wait. mon_lib.cif INFO: link is found (not be used) dist= 1.907 ideal_dist= 1.432 ch:AA res: 8 GLY at:CA .-ch:AA res: 50 TYR at:OH . INFO: link is found (not be used) dist= 2.126 ideal_dist= 1.645 ch:AA res: 13 ARG at:NH1 .-ch:AA res: 54 MET at:SD . INFO: link is found (not be used) dist= 1.820 ideal_dist= 1.462 ch:AA res: 13 ARG at:NH1 .-ch:AA res: 54 MET at:CE . INFO: link is found (not be used) dist= 1.757 ideal_dist= 1.395 ch:AA res: 13 ARG at:NE .-ch:AA res: 119 PHE at:CD1 . INFO: link is found (not be used) dist= 1.572 ideal_dist= 1.462 ch:AA res: 13 ARG at:NH2 .-ch:AA res: 125 LEU at:CD2 . INFO: link is found (not be used) dist= 1.751 ideal_dist= 1.513 ch:AA res: 33 ILE at:CG2 .-ch:AA res: 39 PRO at:CG . INFO: link is found (not be used) dist= 1.451 ideal_dist= 1.513 ch:AA res: 33 ILE at:CG2 .-ch:AA res: 39 PRO at:CD . INFO: link is found (not be used) dist= 1.759 ideal_dist= 1.524 ch:AA res: 58 LYS at:CD .-ch:AA res: 130 LEU at:CB . INFO: link is found (not be used) dist= 1.725 ideal_dist= 1.513 ch:AA res: 58 LYS at:CD .-ch:AA res: 130 LEU at:CD1 . INFO: link is found (not be used) dist= 1.578 ideal_dist= 1.524 ch:AA res: 83 ARG at:CD .-ch:AA res: 101 PRO at:CG . INFO: link is found (not be used) dist= 1.843 ideal_dist= 1.457 ch:AA res: 83 ARG at:NE .-ch:AA res: 101 PRO at:CB . INFO: link is found (not be used) dist= 0.666 ideal_dist= 1.457 ch:AA res: 83 ARG at:NE .-ch:AA res: 101 PRO at:CG . INFO: link is found (not be used) dist= 1.275 ideal_dist= 1.510 ch:AA res: 83 ARG at:CZ .-ch:AA res: 101 PRO at:CB . INFO: link is found (not be used) dist= 1.102 ideal_dist= 1.510 ch:AA res: 83 ARG at:CZ .-ch:AA res: 101 PRO at:CG . INFO: link is found (not be used) dist= 1.191 ideal_dist= 1.457 ch:AA res: 83 ARG at:NH1 .-ch:AA res: 101 PRO at:CA . INFO: link is found (not be used) dist= 0.690 ideal_dist= 1.457
Re: [ccp4bb] co-crystallization
Hi YT- We normally prepare our ligand stocks in DMSO and add this to the protein in 3-fold molar excess. The majority of our ligands are quite insoluble and precipitate when the DMSO concentration decreases upon addition to the protein... so I am not surprised that you are seeing this. If your compound does not bind your protein tightly, you might consider using a 5-fold molar excess of ligand. Some proteins crash out if the protein concentration in high when you add the ligand. For those situations, we complex the ligand with dilute protein (1-2 mg/ml), and then concentrate this for crystallization trials. I have had proteins where we had to complex the dilute protein with ligand, and then let it sit overnight at 4C before we concentrated the protein. We normally incubate the protein+ligand at 4C for 1-3 hours for binding before we set up the crystallization experiments. Another scenario might be addition of ligand to the protein followed by incubation at room temp for ~1hr. Then centrifuge at 4C, keep protein at 4C and set up your trays. Hope this helps! annie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yvonne TAN Yih Wan Sent: Thursday, August 25, 2011 2:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] co-crystallization Hi , I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)? Any suggestions would help. Thanks TY
Re: [ccp4bb] co-crystallization
Dear TY, Typically between 5-10x molar concentration over the protein is enough to ensure binding when the IC50 is uM to low mM. For tighter binding compounds (nM to low uM), 2-5x is sufficient. Whatever you do, when the precipitate occurs DO NOT REMOVE it. I learned to my chagrin that you change all the dynamics of the drop when you do. I ended up with empty crystals until I left the precipitate in place. Think of it this way- Free protein + compound ↔ protein:compound complex + precipitate (mix of protein + compound) If you change the equilibrium by removing the precipitate, you remove the “pressure” on the P:C complex, and it will dissociate to P + C. The precipitate acts as a reserve of protein and compound, thus favoring (or stabilizing) the P:C complex. I set drops up as a slurry frequently, and if I get crystals, they always have the compound bound. Pay attention to the drops if you are screening, because it will be important to note what makes the precipitated solution better (clear drops=solubilizing) or worse (aggregated drops=decreased solubility of your complex). You can also try suspending your compound in LMW PEG’s (200-400 FW) instead of DMSO. Either way, try using DMSO (~20%) or LMW PEG (~30%) depending on your crystallization conditions as a cryoprotectant. Any crystals that grow have some small amount of those agents in them already, so they should be more tolerant of them in higher concentrations. Best of luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yvonne TAN Yih Wan Sent: Thursday, August 25, 2011 2:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] co-crystallization Hi , I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)? Any suggestions would help. Thanks TY -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] merge chains in coot
Calculate - Merge Molecules Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of jab Sent: Thursday, August 25, 2011 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] merge chains in coot Hi I've tried to find where to merge chains in coot, eg merge built co-factor to protein chain, or merge all my ions (Na, Cl, Mg etc) together. Any pointers? Thanks Jim Brannigan
Re: [ccp4bb] Aging PEGs
Time to start digging in the archives. Try looking at work by Fran Jurnak in 1986 (J. Cryst. Growth 76, 577) and Bill Ray's work in 1985 (Analytical Biochem 146, 307), and then the works that cite them. I thought this was common knowledge, but I guess it goes in phases. Aging of poly(oligo)-oxyethylene-based compounds is well known in the surfactant field as it changes the chemical properties of common detergents (Brij, Triton, C10E6, etc.), not only by adding aldehydes and carboxylates to the system, but also by increasing metal binding. It is a sobering sight to see old PEG cross-link your 3-month old crystal: the damn thing wouldn't dissolve after 2 hours in plain buffer, but when I poked it with a glass fiber, the protein oozed out like the center of a cherry cordial, leaving a sad looking deflated shell of a crystal. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Aug 24, 2011, at 5:21 PM, Frank von Delft wrote: And now, does anybody know of systematic data indicating how consistently all this matters? phx On 24/08/2011 21:45, Prince, D Bryan wrote: For those of us truly controlling types :), I used to make the PEG solutions and filter them over a Bio-Rad resin that filtered out all the junk added to stabilize the PEG solution. Then, of course I had to freeze all my PEG solutions in aliquots, or wrap them in foil and store at 4C in the dark. This would take several days, depending on the FW of the PEG. If you are really sensitive about what is in your PEG solutions, try GC-grade PEG's. The FW profile is much more restricted around the reported value (i.e. PEG 3350 molecular biology grade has a broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter peak profile.) Back before you could buy Crystal Screen I, II or HT, you had to make the stock solutions, then make the screen. But at least when you did that, you had all the stocks. Now, I just buy pre-made solutions, and keep them in a drawer with a date opened written on the bottle. Isn't progress grand? :) Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, August 24, 2011 3:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Aging PEGs A while ago I measured the pH's of old and new PEGs and found them very different, and internally attributed all old vs new PEG issues to pH. Upon reflection, this seems too simplistic. Are there other known mechanisms of crystallization capacities of PEGs of various ages? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] loop building with ARP/wARP
Dear Greg, Try ARP/wARP 7.2, released a few days ago, which is better, more stable and should have improved flash of error messages to the log files. ARP/wARP Loopy will not build a 33-residue loop at once, but Classic model building may get the gap shortened. Go to www.arp-warp.org for download. In parallel, from the same page you can submit Classic protein model building for remote execution in Hamburg, which also has ARP/wARP 7.2 installed. Please get back if problems remain. With best regards, Victor Quoting Gregory Bowman gdbow...@jhu.edu: Hi all, I have a large disordered loop (33aa) for a 2.0 Å dataset for which the rest of the structure is well-defined, and phases are decent (Rwork=19.6, Rfree=24.6). I can see some broken up density at one end, but have been unable to convincingly build into into this region manually. I would like to try arp/warp to improve density or possibly extend the loop termini but have been thwarted by some technical problems and would appreciate any advice. I've installed ccp4 6.2.0 on Mac OS X (10.6.8) with fink, both as 32-bit and 64-bit to allow me to run ARP/wARP (version 7.1) from the ccp4i GUI. I've been unable to get loopy to work, and also the standard ARP/wARP classic. When I run ARP/wARP Loops (a.k.a. loopy), I get the message in the log file: Couldn't find any loop to build, loopy will simple copy the pdb file. The program is able to read the sequence .pir file and match it to the structure, and seems from the sequence alignment to identify the regions of the sequence for which there is no structure. I'm guessing that I'm not putting some obvious parameter into the GUI/starting script, but I don't know what that might be. The script generated from the GUI (21_loopy.def) is pasted below. When I try to run ARP/wARP classic for loop building, I get the following message in the logfile: QUITTING ... ARP/wARP module stopped with an error message: REFMAC *** Look for error message in the file: /Users/gbowman/Greg/20_warpNtrace_refine.last.log At the end of the warpNtrace_refine.last.log file, the program reports what seem to be close contacts. Since I don't see this in the structure, and don't have any problems or errors like this when I refine the structure with REMAC, is this from rebuilding, and is this the problem that halts REFMAC? Thanks! Greg = = = = = = 20_warpNtrace_refine.last.log = = = = = = snip Input file :restraints.pdb NUMBER OF MONOMERS IN THE LIBRARY : 11465 with complete description: 11465 NUMBER OF MODIFICATIONS:53 NUMBER OF LINKS:66 I am reading libraries. Please wait. - energy parameters - monomers description (links mod ) BFONT COLOR=#FF!--SUMMARY_BEGIN-- !--SUMMARY_END--/FONT/B Number of atoms:2463 Number of residues : 859 Number of chains : 3 I am reading library. Please wait. mon_lib.cif INFO: link is found (not be used) dist= 1.907 ideal_dist= 1.432 ch:AA res: 8 GLY at:CA .-ch:AA res: 50 TYR at:OH . INFO: link is found (not be used) dist= 2.126 ideal_dist= 1.645 ch:AA res: 13 ARG at:NH1 .-ch:AA res: 54 MET at:SD . INFO: link is found (not be used) dist= 1.820 ideal_dist= 1.462 ch:AA res: 13 ARG at:NH1 .-ch:AA res: 54 MET at:CE . INFO: link is found (not be used) dist= 1.757 ideal_dist= 1.395 ch:AA res: 13 ARG at:NE .-ch:AA res: 119 PHE at:CD1 . INFO: link is found (not be used) dist= 1.572 ideal_dist= 1.462 ch:AA res: 13 ARG at:NH2 .-ch:AA res: 125 LEU at:CD2 . INFO: link is found (not be used) dist= 1.751 ideal_dist= 1.513 ch:AA res: 33 ILE at:CG2 .-ch:AA res: 39 PRO at:CG . INFO: link is found (not be used) dist= 1.451 ideal_dist= 1.513 ch:AA res: 33 ILE at:CG2 .-ch:AA res: 39 PRO at:CD . INFO: link is found (not be used) dist= 1.759 ideal_dist= 1.524 ch:AA res: 58 LYS at:CD .-ch:AA res: 130 LEU at:CB . INFO: link is found (not be used) dist= 1.725 ideal_dist= 1.513 ch:AA res: 58 LYS at:CD .-ch:AA res: 130 LEU at:CD1 . INFO: link is found (not be used) dist= 1.578 ideal_dist= 1.524 ch:AA res: 83 ARG at:CD .-ch:AA res: 101 PRO at:CG . INFO: link is found (not be used) dist= 1.843 ideal_dist= 1.457 ch:AA res: 83 ARG at:NE .-ch:AA res: 101 PRO at:CB . INFO: link is found (not be used) dist= 0.666 ideal_dist= 1.457 ch:AA
[ccp4bb] 2nd MX User Workshop at Diamond - September 7th-8th, 2011
Dear All I quick reminder - last chance to sign up for the 2nd MX User Workshop at Diamond - the workshop is part of the Synchrotron User Meeting, September 7th-8th, 2011. Workshop sessions include presentations from staff and users on latest developments, spectroscopy, crystal dehydration and room temperature data collection, using microfocus beam and how to get the most from your beamtime. Workshop info: http://www.diamond.ac.uk/Home/Events/Synchrotron_User_Meeting_2011/MX.html Registration: http://www.diamond.ac.uk/Home/Events/Synchrotron_User_Meeting_2011/Registration-system.html Best wishes Thomas Sorensen Principal Beamline Scientist Macromolecular Crystallography Diamond Light Source
[ccp4bb] Fwd: Re: [ccp4bb] co-crystallization
Successful complexation depends on the concentration of protein, ligand, and the Kd of the protein-ligand complex. For Kd[protein], you will probably require [ligand] 10 x Kd. As Kd approaches [protein], slightly superstoichiometric quantities will be sufficient for full occupancy. For Kd [protein], stoichiometric quantities of ligand will suffice. Basically you need a [ligand] that puts near saturation on the binding isotherm. Cheers. On 8/25/2011 2:03 AM, Yvonne TAN Yih Wan wrote: Hi , I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)? Any suggestions would help. Thanks TY -- 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
[ccp4bb] Web Seminar: An Insider's Guide to CrystalClear and d*TREK: Improve Your Results with these New Features and Tips
Dear colleagues, I would like to draw your attention to an upcoming free, educational webinar to be presented by Jim Pflugrath, Ph. D. titled An Insider's Guide to CrystalClear and d*TREK: Improve Your Results with these New Features and Tips. In this webinar, Jim will discuss three important new features or tips for using CrystalClear and d*TREK which will lead to better results: lower Rmeas and higher I/sigmaI. New input parameters for finding spots is one new feature. Another feature is a better way perform positional refinement before and during Bragg reflection integration. A third tip is how to improve your scaling results by changing the input parameters to dtscaleaverage. These tips and others will give you better results with CrystalClear. This webinar is scheduled to occur on Thursday, September 1st 10:00 AM CDT (8:00 AM PDT / 4:00 PM GMT). You can find more information, including a registration link at: http://www.rigaku.com/protein/webinars.html. Best regards, Angela Criswell -- Angela R. Criswell, Ph. D. VP, Life Sciences Rigaku Americas Corporation 9009 New Trails Drive The Woodlands, TX 77381 USA Ph: +1 281 362 2300 ext. 216 Fax: +1 281 364 3628 Email: angela.crisw...@rigaku.com URL: http://www.rigaku.com
Re: [ccp4bb] number of cycles in refmac
Hi Ian (Yes, your technical point about semantics is correct, I meant over-fitting.) To pin down your points, though, you're saying: 1) Don't use Rfree, instead look at LLfree or Hamilton Rfree. 2) Compare only the final values at convergence when choosing between different parametrizations (=models) Point #1 - fair point; the reason Rfree is popular, though, is because it is a /relative/ metric, i.e. by now we have a sense of what good is. So I predict an uphill fight for LLfree. Point #2 would hold if we routinely let our refinements run to convergence; seems common though to run 10 cycles or 50 cycles instead and draw conclusions from the behaviour of the metrics. Are the conclusions really much different from the comparison-at-convergence you advocate? Which is in practice often less convenient. Cheers phx Isn't this the purpose of cross-validation, to use an independent measure to judge when the refinement is /not/ producing the best model? If the value of your chosen X-validation metric at convergence indicates a problem with the model, parameterisation, weighting etc then clearly the target function is not indeed the final word: the solution is to fix whatever is wrong and do the refinement again, until you get a satisfactory value for your metric. This may be true; but as it is independent of refinement, is it not nevertheless the only measure I should trust? No there several possible functions of the test set (e.g. Hamilton Rfree, LLfree) that you could use, all potentially equally valid X-validation metrics. I would have more faith in a function such as LLfree in which the contributions of the reflections are at least weighted according to their reliability. It just seems bizarre that important decisions are being based on measurements that may have gross errors without taking those errors into account. Or maybe what you intended to say: only trust refinements for which Rfree decreases monotonically, because only then do you have a valid choice of parameters. No, as I indicated above, what Rfree does before convergence is attained is totally meaningless, only the value obtained _at_ convergence is meaningful as a X-validation statistic. We wouldn't be having this discussion if the refinement program omitted the meaningless intermediate values and only printed out the final Rfree or LLfree. I'm saying that Rfree is not the best X-validation metric because poorly measured data are not properly weighted: this is what Acta paper I referenced is saying. Cheers -- Ian
Re: [ccp4bb] number of cycles in refmac
Point #2 would hold if we routinely let our refinements run to convergence; seems common though to run 10 cycles or 50 cycles instead and draw conclusions from the behaviour of the metrics. Are the conclusions really much different from the comparison-at-convergence you advocate? Which is in practice often less convenient. May be run refinement many times with slightly different starting points and consider looking at an ensemble? Convergence seems to have a vague meaning when it comes to macromolecular crystallographic structure refinement (p. 75-76 here: http://www.phenix-online.org/presentations/latest/pavel_refinement_general.pdf ) Pavel
