[ccp4bb] WM_DELETE_WINDOW
Who do I contact to spread the news that you can bind the WM_DELETE_WINDOW protocol of the main ccp4i window to the same callback as the EXIT button in the lower right hand corner of the window? Translation: will prevent annoying abrupt quitting behavior upon accidentally closing main ccp4i window. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
Re: [ccp4bb] WM_DELETE_WINDOW
Maybe it is bound on second look--But it does not confirm exit, which would be handy. On Mar 4, 2008, at 4:46 AM, James Stroud wrote: Who do I contact to spread the news that you can bind the WM_DELETE_WINDOW protocol of the main ccp4i window to the same callback as the EXIT button in the lower right hand corner of the window? Translation: will prevent annoying abrupt quitting behavior upon accidentally closing main ccp4i window. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
Re: [ccp4bb] WM_DELETE_WINDOW
Dear James Generally the best people to contact about this sort of news are the CCP4 staff at Daresbury, [EMAIL PROTECTED] I'm sure that I've missed something, however - both the Exit button and the window close operation on the main window (which I understood to be bound to WM_DELETE_WINDOW, see browser_utils.tcl) are already bound to the same ExitInterface procedure (defined in taskwindow.tcl). This procedure saves the state of the interface and then exits, destroying both the main CCP4i window and all children windows without prompting first. So I'm not sure what it is that you want. Could you clarify further? Best wishes Peter James Stroud wrote: Who do I contact to spread the news that you can bind the WM_DELETE_WINDOW protocol of the main ccp4i window to the same callback as the EXIT button in the lower right hand corner of the window? Translation: will prevent annoying abrupt quitting behavior upon accidentally closing main ccp4i window. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com -- ___ Peter J Briggs, [EMAIL PROTECTED] Tel: +44 1925 603826 CCP4, [EMAIL PROTECTED] Fax: +44 1925 603825 http://www.ccp4.ac.uk/ Daresbury Laboratory, Daresbury, Warrington WA4 4AD
Re: [ccp4bb] WM_DELETE_WINDOW
Having just sampled a few Linux apps, the most common behaviour for the close decoration seems to be to close without warning if there is no unsaved work, or to close with warning otherwise. At first glance, the CCP4i main window never has an unsaved state, so I was going to disagree with you. However, if you have a task window open (which may have unsaved program parameters entered in it), then closing the main window also closes the task window, losing the info you were entering. So I would vote in favour of a confirm option. Or even better, close automatically if no other windows are open, or ask for confirmation if there are any other windows open (but the benefit of this refinement is marginal - only worth doing if it is really easy). Kevin James Stroud wrote: Maybe it is bound on second look--But it does not confirm exit, which would be handy. On Mar 4, 2008, at 4:46 AM, James Stroud wrote: Who do I contact to spread the news that you can bind the WM_DELETE_WINDOW protocol of the main ccp4i window to the same callback as the EXIT button in the lower right hand corner of the window? Translation: will prevent annoying abrupt quitting behavior upon accidentally closing main ccp4i window. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
Re: [ccp4bb] WM_DELETE_WINDOW
I'm not sure I agree with having a confirm button. This would try to address a 'problem' in a way that has us all clicking a 'confirm' button every time we close the interface, to stop accidentally closing the interface every now and again. As closing an interface (plus children) window represents a minor loss of work (just a few clicks very often), I would argue that consistency with other applications is less important than the irritation of another confirm button. I'm surely not the only one who grumbles 'yes of course I wanted to exit, why else do you think I clicked the exit button' ;-) Just my opinion.. Johan Kevin Cowtan wrote: Having just sampled a few Linux apps, the most common behaviour for the close decoration seems to be to close without warning if there is no unsaved work, or to close with warning otherwise. At first glance, the CCP4i main window never has an unsaved state, so I was going to disagree with you. However, if you have a task window open (which may have unsaved program parameters entered in it), then closing the main window also closes the task window, losing the info you were entering. So I would vote in favour of a confirm option. Or even better, close automatically if no other windows are open, or ask for confirmation if there are any other windows open (but the benefit of this refinement is marginal - only worth doing if it is really easy). Kevin James Stroud wrote: Maybe it is bound on second look--But it does not confirm exit, which would be handy. On Mar 4, 2008, at 4:46 AM, James Stroud wrote: Who do I contact to spread the news that you can bind the WM_DELETE_WINDOW protocol of the main ccp4i window to the same callback as the EXIT button in the lower right hand corner of the window? Translation: will prevent annoying abrupt quitting behavior upon accidentally closing main ccp4i window. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com -- + Dr. Johan P. Turkenburg X-ray facilities manager York Structural Biology Laboratory University of York Phone (+) 44 1904 328251 York YO10 5DD UK Fax (+) 44 1904 328266 +
[ccp4bb] phenix.refine and refmac
Dear All, I have post a similar question about CNS and refmac before, now in another structure I met a similar problem. I have an almost finished structure, the Rfree of which is about 0.28 by refmac. Then I used phenix to refine it, below is the result: REMARK REFINEMENT SUMMARY: QUICK FACTS *** REMARK Start: r_work = 0.1970 r_free = 0.2892 bonds = 0.006 angles = 1.213 REMARK Final: r_work = 0.1917 r_free = 0.2617 bonds = 0.008 angles = 1.374 REMARK Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file. But I found that at the first several cycle of refmac the Rfree decreased, then both the R and Rfree values continued increasing and FOM decreasing. The best R/Rfree/FOM during the refinement is - Overall R factor = 0.1932 Free R factor= 0.2513 Overall figure of merit = 0.8168 - And after 40 cycles the final result is: - Overall R factor = 0.2008 Free R factor= 0.2772 Overall figure of merit = 0.7902 - The values looks like keep going up if increase the cycles. Then which value should I take as the final result? The phenix or the best Refmac result or I have to take a converged value from refmac?
Re: [ccp4bb] phenix.refine and refmac
phenix.refine also produces an mtz file by default, and that can be auto-opened with coot, along with the coordinates. On Mar 4, 2008, at 7:27 AM, yang li wrote: Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file.
Re: [ccp4bb] Crosslinking reagents
I will try that. But I don't think people can tell the final concentration of glutaraldehyde in the drop by using its vapor. Maybe using direct soaking is what I should do. By the way, should I quench it by ammonium or not? Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 On Sun, Mar 2, 2008 at 12:28 AM, Nian Huang [EMAIL PROTECTED] wrote: Dear all, I know most people use glutaraldehyde as the crosslinking reagent for their crystals. But there are a lot of other crosslinking chemicals available both in protein chemistry and histology, such as simple formaldehyde. Did anybody have experience with other chemicals and how did they compared with glutaradehyde? I have a crystal very sensitive to glutaradehyde but I really wants to try the crosslinking method. Thanks, Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390
Re: [ccp4bb] phenix.refine and refmac
Yes, I got the mtz file which can be oopened by coot, so can I take the phenix result? I am customed to use refmac before, but now is confused when several choices come out. On 3/4/08, William Scott [EMAIL PROTECTED] wrote: phenix.refine also produces an mtz file by default, and that can be auto-opened with coot, along with the coordinates. On Mar 4, 2008, at 7:27 AM, yang li wrote: Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file.
Re: [ccp4bb] Crosslinking reagents
Nian, It is also important to point out that gluteraldehyde is quite penetrating (which is why it is used as a fixative in EM) and volitile. A 1% solution is 100 mM, which is quite concentrated. Adding gluteraldehyde by vapor diffusion is quite effective and gentle, but does take a bit of time (hours). Remember that gluteraldehyde fixes by creating Schiff's bases. Thus, a non- colored crystal turns a light gold color, which is the best optical indicator of fixation. However, things will go awry if there are lots of solutes around with free amines. The best example is Tris buffer. So soak/grow your in mother liquor with no free amines other that those on the protein. You can quench it with ammonium chloride if desired. Good luck, 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: [EMAIL PROTECTED] On Mar 4, 2008, at 11:13 AM, David M Shechner wrote: Quoting Nian Huang [EMAIL PROTECTED]: I will try that. But I don't think people can tell the final concentration of glutaraldehyde in the drop by using its vapor. Maybe using direct soaking is what I should do. By the way, should I quench it by ammonium or not? Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 Often it's the case that explicit knowledge of the gluteraldehyde concentration in the vapour isn't as important as empirical determination of the exposure time you subject the drop to, prior to moving it to a new well of non-crosslinking mother liquor. Here's the first (to my knowledge) reference that uses this technique; they mention using a time-course to determine when to quench: Lusty, CJ (1999) A gentle vapor-diffusion technique for cross- linking of protein crystals for cryocrystallography. J. Appl. Cryst. 32. 106-112. Cheers, d.s. === David M. Shechner, Graduate Student Bartel Laboratory Department of Biology Whitehead Institute for Biomedical Research Massachusetts Institute of Technology [EMAIL PROTECTED] ===
Re: [ccp4bb] Crosslinking reagents
Quoting Nian Huang [EMAIL PROTECTED]: I will try that. But I don't think people can tell the final concentration of glutaraldehyde in the drop by using its vapor. Maybe using direct soaking is what I should do. By the way, should I quench it by ammonium or not? Nian Huang Dept of Biochemistry UT Southwestern Medical Center Dallas, TX 75390 Often it's the case that explicit knowledge of the gluteraldehyde concentration in the vapour isn't as important as empirical determination of the exposure time you subject the drop to, prior to moving it to a new well of non-crosslinking mother liquor. Here's the first (to my knowledge) reference that uses this technique; they mention using a time-course to determine when to quench: Lusty, CJ (1999) A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography. J. Appl. Cryst. 32. 106-112. Cheers, d.s. === David M. Shechner, Graduate Student Bartel Laboratory Department of Biology Whitehead Institute for Biomedical Research Massachusetts Institute of Technology [EMAIL PROTECTED] ===
Re: [ccp4bb] phenix.refine and refmac
Dear Yang, it would be better if you could provide more details about your refinement strategies. As I can see from your results you might have used the default values in phenix giving you finally very low rmsd values. Refmac by default is not using so tight restraints and even if you used tight restraints in refmac the difference between R and Rfree might be much lower than the 7% in Phenix. I am guessing that the values that you are reporting have to do also with the weight values each program uses and how you are define them. Also have you used TLS in any of the two programs? regards Nikos - Dr. Nikos Pinotsis Section of Structural Biology Institute of Cancer Research Chester Beatty Laboratories 237 Fulham Road London SW3 6JB, UK Tel: +44 20 7153 5453 / 5447 - yang li wrote: Dear All, I have post a similar question about CNS and refmac before, now in another structure I met a similar problem. I have an almost finished structure, the Rfree of which is about 0.28 by refmac. Then I used phenix to refine it, below is the result: REMARK REFINEMENT SUMMARY: QUICK FACTS *** REMARK Start: r_work = 0.1970 r_free = 0.2892 bonds = 0.006 angles = 1.213 REMARK Final: r_work = 0.1917 r_free = 0.2617 bonds = 0.008 angles = 1.374 REMARK Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file. But I found that at the first several cycle of refmac the Rfree decreased, then both the R and Rfree values continued increasing and FOM decreasing. The best R/Rfree/FOM during the refinement is - Overall R factor = 0.1932 Free R factor= 0.2513 Overall figure of merit = 0.8168 - And after 40 cycles the final result is: - Overall R factor = 0.2008 Free R factor= 0.2772 Overall figure of merit = 0.7902 - The values looks like keep going up if increase the cycles. Then which value should I take as the final result? The phenix or the best Refmac result or I have to take a converged value from refmac? The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] WM_DELETE_WINDOW
On Mar 4, 2008, at 5:31 AM, Johan Turkenburg wrote: I would argue that consistency with other applications is less important than the irritation of another confirm button. I'm surely not the only one who grumbles 'yes of course I wanted to exit, why else do you think I clicked the exit button' ;-) First, the close button could map to a different call back than the EXIT button. Intercepting and putting up a confirmation dialog is trivial and amounts, inclusively, to (1) changing the already existing binding self.winfo_toplevel().protocol(WM_DELETE_WINDOW, self.wm_delete) plus (2) three new lines of code: def wm_delete(self): if tkMessageBox.askyesno(Exit, Exit ccp4i?): self.exit() Second, I think the behavior most consistent with almost every application created in the last 15 years is to ask to close if the user has made unsaved changes. This could be as simple as adding a callback to the input fields when they lose focus (which I assume are already sub-classed any way). Consider, for example, one's trying to import a CNS file, where one spends 25 minutes getting the 6x,F10.5,6x,I10,etc. just right, and after the 5th iteration one tries to close the task input window only to close the main ccp4i window, which exits without so much as a core- dump. For my 4 function calculator, I don't want it to ask me if I want it to close. For my word processor, I expect confirmation if I've made changes. For a crystallography suite, I would err on the side of caution. James -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
[ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Hi Stephen, Fun question! A quick google search with structure based sequence alignment gave me this site which looks promising: http://www.cgl.ucsf.edu/home/meng/grpmt/structalign-content.html Let me know how it turns out! Sophia On Tue, Mar 4, 2008 at 11:13 AM, Stephen Graham [EMAIL PROTECTED] wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] phenix.refine and refmac
Hi Yang how many reflections do you have in your test-set for calculating R-free? Too few reflections, typically less than 500, may not constitute a statistically robust cross-validation data set, and thus may lead to fluctuations in R-free plus a tendency for R-free to increase as a function of refinement cycle. Some of the early publications from Axel Brunger on crystallographic cross-validation address the need for enough reflections (500) in the test-set. In addition, the presence of even a handful of strong but inaccurately measured/integrated low-resolution reflections in a limited test-set can aggravate abnormal behavior in R-free. Best wishes Savvas toQuoting yang li [EMAIL PROTECTED]: Dear All, I have post a similar question about CNS and refmac before, now in another structure I met a similar problem. I have an almost finished structure, the Rfree of which is about 0.28 by refmac. Then I used phenix to refine it, below is the result: REMARK REFINEMENT SUMMARY: QUICK FACTS *** REMARK Start: r_work = 0.1970 r_free = 0.2892 bonds = 0.006 angles = 1.213 REMARK Final: r_work = 0.1917 r_free = 0.2617 bonds = 0.008 angles = 1.374 REMARK Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file. But I found that at the first several cycle of refmac the Rfree decreased, then both the R and Rfree values continued increasing and FOM decreasing. The best R/Rfree/FOM during the refinement is - Overall R factor = 0.1932 Free R factor= 0.2513 Overall figure of merit = 0.8168 - And after 40 cycles the final result is: - Overall R factor = 0.2008 Free R factor= 0.2772 Overall figure of merit = 0.7902 - The values looks like keep going up if increase the cycles. Then which value should I take as the final result? The phenix or the best Refmac result or I have to take a converged value from refmac?
Re: [ccp4bb] phenix.refine and refmac
One point which I don't understand is how can someone compare the two different programs when they don't use the same numbers for xray:geometry terms? Taking the default settings for a given resolution might not be enough.. ! On Tue, Mar 4, 2008 at 7:58 PM, Savvas Savvides [EMAIL PROTECTED] wrote: Hi Yang how many reflections do you have in your test-set for calculating R-free? Too few reflections, typically less than 500, may not constitute a statistically robust cross-validation data set, and thus may lead to fluctuations in R-free plus a tendency for R-free to increase as a function of refinement cycle. Some of the early publications from Axel Brunger on crystallographic cross-validation address the need for enough reflections (500) in the test-set. In addition, the presence of even a handful of strong but inaccurately measured/integrated low-resolution reflections in a limited test-set can aggravate abnormal behavior in R-free. Best wishes Savvas toQuoting yang li [EMAIL PROTECTED]: Dear All, I have post a similar question about CNS and refmac before, now in another structure I met a similar problem. I have an almost finished structure, the Rfree of which is about 0.28 by refmac. Then I used phenix to refine it, below is the result: REMARK REFINEMENT SUMMARY: QUICK FACTS *** REMARK Start: r_work = 0.1970 r_free = 0.2892 bonds = 0.006 angles = 1.213 REMARK Final: r_work = 0.1917 r_free = 0.2617 bonds = 0.008 angles = 1.374 REMARK Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file. But I found that at the first several cycle of refmac the Rfree decreased, then both the R and Rfree values continued increasing and FOM decreasing. The best R/Rfree/FOM during the refinement is - Overall R factor = 0.1932 Free R factor= 0.2513 Overall figure of merit = 0.8168 - And after 40 cycles the final result is: - Overall R factor = 0.2008 Free R factor= 0.2772 Overall figure of merit = 0.7902 - The values looks like keep going up if increase the cycles. Then which value should I take as the final result? The phenix or the best Refmac result or I have to take a converged value from refmac? -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
On Tuesday 04 March 2008 11:13, Stephen Graham wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. My preference is for the CE-MC server. There are several sites that offer this, or you can install it locally. Here's one: bioinformatics.albany.edu/~cemc Another one is Tcoffee: http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi When I've done head-to-head comparisons, I have found the output from CE-MC to be preferable. In particular, in cases where you have two structures A and B that have the same length but have an area in which the structures differ, CE-MC will favor the alignment AAaaa... BB...bbb whereas Tcoffee will report AAaaa BBbbb For my purposes, the first is correct, even if aaa and bbb are sequence-homologous. -- Ethan A MerrittCourier Deliveries: 1959 NE Pacific Dept of Biochemistry Health Sciences Building University of Washington - Seattle WA 98195-7742
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
What about Dennis Madsen et al's Indonesia? http://xray.bmc.uu.se/dennis/ I don't think it's been updated in years (or I have a old link), but it's still quite nice as it runs locally using java. AGS Date: Tue, 4 Mar 2008 12:02:20 -0800 From: [EMAIL PROTECTED] Subject: Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments To: CCP4BB@JISCMAIL.AC.UK On Tuesday 04 March 2008 11:13, Stephen Graham wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. My preference is for the CE-MC server. There are several sites that offer this, or you can install it locally. Here's one: bioinformatics.albany.edu/~cemc Another one is Tcoffee: http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi When I've done head-to-head comparisons, I have found the output from CE-MC to be preferable. In particular, in cases where you have two structures A and B that have the same length but have an area in which the structures differ, CE-MC will favor the alignment AAaaa... BB...bbb whereas Tcoffee will report AAaaa BBbbb For my purposes, the first is correct, even if aaa and bbb are sequence-homologous. -- Ethan A MerrittCourier Deliveries: 1959 NE Pacific Dept of Biochemistry Health Sciences Building University of Washington - Seattle WA 98195-7742 _ Need to know the score, the latest news, or you need your Hotmail®-get your fix. http://www.msnmobilefix.com/Default.aspx
[ccp4bb] Removal of glycosylation sites in Picha expression construct
Dear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
This is not entirely uncommon. Did you try removing just one of the two sites (sometimes it helps) - combined with enzymatic deglycosylation this may give you good enough protein to work with. If you try other hosts, I would definitely consider Schisosaccharomyces pombe - its glycosylation patterns are much simpler. Other likely candidates include regular bakers' yeast and Hansenula polymorpha. I would try both the native and the mutant proteins in those hosts. There are tricks one can play with Pichia to try and make it express the deglycosylated protein but it almost sounds like you've hit on something very essential since you're going from good expression down to no expression. Lastly, you could try insect cells :) Artem Dear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Hi Stephen, I will add to the previously mentioned ones the MSD ssm server at EBI? http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver You also have the Godzik tools if your proteins have flexible regions. FATCAT http://fatcat.burnham.org/ and POSA http://fatcat.burnham.org/POSA/ Good luck, Joao João M. Dias Ollmann Saphire Lab The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA On Mar 4, 2008, at 11:13 AM, Stephen Graham wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
I can recommend MUSTANG: http://p2p.cs.mu.oz.au/mustang/ Web server version is http://p2p.cs.mu.oz.au/mustang/php/ paper is here: http://www.ncbi.nlm.nih.gov/pubmed/16736488?ordinalpos=5itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Ashley On 05/03/2008, at 6:13 AM, Stephen Graham wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549 Ashley Buckle Ph.D NHMRC Senior Research Fellow The Department of Biochemistry and Molecular Biology School of Biomedical Sciences, Faculty of Medicine Victorian Bioinformatics Consortium (VBC) Monash University, Clayton, Vic 3800 Australia http://www.med.monash.edu.au/biochem/staff/abuckle.html iChat/AIM: blindcaptaincat skype: ashley.buckle Tel: (613) 9902 0269 (office) Tel: (613) 9905 1653 (lab) Fax : (613) 9905 4699
Re: [ccp4bb] phenix.refine and refmac
Thanks for your replies. I did use default settings in phenix refine, included TLS in it. This is an about 2.3A data, and the number Rfree used to refine is big enough. I also kept the same Rfree in two programs. In refmac I also tried diffenrent weighting sets--from defaut 0.3 to 0.02, TLS included--maybe there are some other ways to define the restrains. The R and Rfree will increase as the weighting is set too low, and the gap didnot improve much. I donnot know in this case if the phenix refine has converged, but the not stable Rfree in refmac made me nervous. Simply, in this case, which one should I choose? Certainly to my private opinion I would prefer to Scott's answer, Rfree is the most obvious parameter than something esle like geometry, eg. Just looks good. :) On 3/5/08, Partha Chakrabarti [EMAIL PROTECTED] wrote: One point which I don't understand is how can someone compare the two different programs when they don't use the same numbers for xray:geometry terms? Taking the default settings for a given resolution might not be enough.. ! On Tue, Mar 4, 2008 at 7:58 PM, Savvas Savvides [EMAIL PROTECTED] wrote: Hi Yang how many reflections do you have in your test-set for calculating R-free? Too few reflections, typically less than 500, may not constitute a statistically robust cross-validation data set, and thus may lead to fluctuations in R-free plus a tendency for R-free to increase as a function of refinement cycle. Some of the early publications from Axel Brunger on crystallographic cross-validation address the need for enough reflections (500) in the test-set. In addition, the presence of even a handful of strong but inaccurately measured/integrated low-resolution reflections in a limited test-set can aggravate abnormal behavior in R-free. Best wishes Savvas toQuoting yang li [EMAIL PROTECTED]: Dear All, I have post a similar question about CNS and refmac before, now in another structure I met a similar problem. I have an almost finished structure, the Rfree of which is about 0.28 by refmac. Then I used phenix to refine it, below is the result: REMARK REFINEMENT SUMMARY: QUICK FACTS *** REMARK Start: r_work = 0.1970 r_free = 0.2892 bonds = 0.006 angles = 1.213 REMARK Final: r_work = 0.1917 r_free = 0.2617 bonds = 0.008 angles = 1.374 REMARK Since the map from phenix couldnot be opened by coot directly--or I donnot know how to--I used refmac to get a mtz map file. But I found that at the first several cycle of refmac the Rfree decreased, then both the R and Rfree values continued increasing and FOM decreasing. The best R/Rfree/FOM during the refinement is - Overall R factor = 0.1932 Free R factor= 0.2513 Overall figure of merit = 0.8168 - And after 40 cycles the final result is: - Overall R factor = 0.2008 Free R factor= 0.2772 Overall figure of merit = 0.7902 - The values looks like keep going up if increase the cycles. Then which value should I take as the final result? The phenix or the best Refmac result or I have to take a converged value from refmac? -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515
Re: [ccp4bb] protein expression
Hi Chen, In this case, it seems that linker region is of great importance for the proper folding of the two linked domains. I have not much experience in linker region design, generally use (GS)5-10 times. However it depends on individual case. Anyone who has successful experience in linker region design might share your success with others. Thanks a lot. Regards, Donghui On 3/5/08, Daniel Jin [EMAIL PROTECTED] wrote: Hi, I have a protein with two independently folded domains. I can express either one in bacteria with pretty good expression yield. However, when I put them together with a linker, the expression drops significantly. I can barely see any soluble protein and most of it is now inclusion bodies. I used the same bacteria strain and expression/purification conditions. Is it normal? Any suggestion about how to improve? Many thanks. Best, Chen -- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.http://us.rd.yahoo.com/evt=51733/*http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ
[ccp4bb] Problem with DM
Dear Crystallographers, I failed to run DM in ccp4 package recently and got the errors as below: a href=http://www.yorvic.york.ac.uk/~cowtan/dm/refs.html;dm reference:/a blockquote K. Cowtan (1994), dm: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, 31, p34-38. /blockquotep a name=tocdmh2Contents/h2/a ul lia href=#commanddmCommand input/a lia href=#commentsdmComments/a lia href=#mtzindmMTZ input/a lia href=#datachkdmData Checking/a lia href=#datascldmData Scaling/a lia href=#solmskdmSolvent Mask/a lia href=#cyc0001dmFirst Cycle/a lia href=#dataoutdmOutput/a /ul a name=commanddmh2Command Input/h2/a pre Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#modeMODE/a SOLV Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#combineCOMBINE /a PERT Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#schemeSCHEME /a ALL Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#ncycleNCYCLE /a AUTO Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#solcSOLCONT /a *** Warning Real sub-argument expected Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#ncsmaskNCSMASK /a Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#labinLABIN /a FP=F_p2 SIGFP=SIGF_p2 PHIO=PHIC FOMO=FOM Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#laboutLABOUT /a FDM=FDM PHIDM=PHIDM FOMDM=FOMDM dm: Input error (see above) Times: User: 0.0s System:0.0s Elapsed: 0:00 /pre /html *** * Information from CCP4Interface script *** The program run with command: dm HKLIN /Users/Research/CNS/newtls_refmac4.mtz HKLOUT /Users/Research/CNS/newtls_dm1.mtz SOLOUT /Users/Research/CNS/newtls_dm1.msk has failed with error message dm: Input error (see above) *** #CCP4I TERMINATION STATUS 0 dm: Input error (see above) #CCP4I TERMINATION TIME 04 Mar 2008 22:15:09 #CCP4I MESSAGE Task failed I can not fix it. Can anyone give me some help? Thanks very much!
[ccp4bb] isopropanol as a precipitant
Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.Theinteresting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
There are many cases in literature where the removal of N-linked glycosylations sites reduced the expression levels of secreted proteins. I would recommend removal of the N-linked glycans by enzymes (endo H, PNGase F, etc.) using the wild-type proteins you have obtained and then run a gel or mass spec to determine the M.W. after deglycosylation. Alternatively, you may just leave the glycans on if the mass from glycans are just several thousand daltons (how large is your protein?). Many crystal structures of secreted proteins are solved with the glycans on. They seem to help protein folding in your case.Holly Jing Date: Tue, 4 Mar 2008 16:54:00 -0500 From: [EMAIL PROTECTED] Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct To: CCP4BB@JISCMAIL.AC.UK This is not entirely uncommon. Did you try removing just one of the two sites (sometimes it helps) - combined with enzymatic deglycosylation this may give you good enough protein to work with. If you try other hosts, I would definitely consider Schisosaccharomyces pombe - its glycosylation patterns are much simpler. Other likely candidates include regular bakers' yeast and Hansenula polymorpha. I would try both the native and the mutant proteins in those hosts. There are tricks one can play with Pichia to try and make it express the deglycosylated protein but it almost sounds like you've hit on something very essential since you're going from good expression down to no expression. Lastly, you could try insect cells :) ArtemDear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) _ Connect and share in new ways with Windows Live. http://www.windowslive.com/share.html?ocid=TXT_TAGHM_Wave2_sharelife_012008
