Re: [ccp4bb] salt sensitive complex
Hi Jerry, to summarise your problem, using (close to) physiological buffer, SPR and ITC give you different results, you get different results in different salt strengths and to add to your misery, the proteins precipitate at low salt concentrations when mixed to together. Ok. Given the above, your interaction probably has a large electrostatic component, and this is why the Ks are salt sensitive. This is also possibly why you are getting precipitate. If you start with two happy proteins, and then titrate them in together, the (presumably) complementary electrostatic binding surfaces will interact and cancel each other out, reducing the overall charge on the complex. If the complex is less charged, it will be less soluble, therefore, the complex crashes out (in 1:1 stoich). IMHO, ITC is the more elegant experiment. Ok, so it uses *more* material, but you aren't relying on binding surfaces to nail things to, and you are directly(ish) measuring the heat of the interaction. If, in an ITC cell, two proteins come together and are insoluble, they are able to precipitate. On an SPR chip, they are already immobilised to a surface, and so you wouldn't necessarily detect precipitation forming, also, as you run SPR at much lower concentrations of protein you might not induce precipitation if it is protein concentration dependent. If I see precipitate in my sample after an ITC experiment, I'm always weary of it - but at least I'm aware of it. I would always choose to run an ITC experiment over an SPR (ideally both), but sometimes, ITCs requirements for higher concentration means that it isn't always feasible. In this case, the solubility limit of your complex may prevent you getting good ITC data. I would try and keep buffers consistent between your experiments (stick to physiological salt strength - less awkward reviewer questions), and try repeating your experiments at different pHs. I've had complexes that precipitate at pH 7.5 9.5, but are nice and happy at pH 5.5. Hope this (rather lengthy reply) helps! Dave On 23/01/2008, Jerry McCully [EMAIL PROTECTED] wrote: Dear All: Recently I am pursuing the crystallziation of a complex formd by two individual proteins and I met several interesting problems though they are kind of off-topic. Any suggestions for these problems will be highly appreciated. BIAcore showed about submicromolar affinity(both Kinetic and steady-state fitting) for these two proteins in the complex. However, precipitates immediately appeared when these two proteins were mixed together even at 10uM(0.3mg/ml) concentration in the condition of low salt(less than 20mM NaCl). By the way, these two proteins completely precipitated when the molar ratio is 1:1 in this condition. THerefore, I increased the salt concentraion step by step and finally I can keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM NaCl(the minimum of salt concentration). Wierd thing happened when ITC experiments were carried out to confirm the binding affinity. 20uM in the sample cell and 200uM in the syringe could not give enough heat for a good curve fitting. The optimistic estimation of the affinity is lower than 5uM, which is much lower than the affinity given by BIAcore in the same buffer(25mM Tris plus 150mM NaCl). Now I am suspecting the capability of the interaction between these two proteins. However, I can not explain why these two guys precipitated stoichiometrically if they do not interact with each other. Is the complex salt-sensitive therefore there was just minor binding in the high-salt condition revealed by ITC? I am planning to do the ITC again in the condition of 25mMTris and 60mM NaCl. What if the affinity given by ITC is still much lower than that by BIAcore. Which one should I choose to believe? Are there some better ways that I can validate the binding affinity? Thanks again for your great ideas. Jerry McCully Need to know the score, the latest news, or you need your Hotmail(R)-get your fix. Check it out. -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] Characterization of common salt crystal forms?
Hi Joe, I've known most salt crystals in Phosphate - and I think most people are weary of phosphate. Also, Calcium Sulphate is a fairly common one, esp if your buffers are titrated with sulphuric acid. Fluoride Ions are also prone to form salt crystals with transition metal ions. HTH, Dave On 22/01/2008, Joe Krahn [EMAIL PROTECTED] wrote: Salt crystals are common in macromolecular crystallography. Has anyone tried to tabulate salt crystal forms that commonly occur? I just identified a salt crystal as Mirabilite, made of Na2SO4·10H2O. The high water content makes them rather soft, and may not be recognized as salt right away. In this case, it probably happened because the buffer was made with Na·Citrate + HCl instead of citric acid, while trying to optimize conditions. So, characterization of salt crystals can help to avoid the conditions that cause them. There is probably a reasonably small number of salt crystal forms that are very common in crystallization trials. Maybe it would be useful to tabulate common salt crystals to help guide optimization experiments. Has anyone else tried to use salt crystal information beyond ensuring that it is not protein? Joe Krahn -- David C. Briggs PhD Father Crystallographer http://www.dbriggs.talktalk.net AIM ID: dbassophile
Re: [ccp4bb] microsoft 3-button wheel mouse with OS X 10.5
Dear Bill William Scott wrote Aqua simply behaves by slightly different rules. Although I am a slobbering OS X fan, this lack of customizability to me, as well as a lack of focus-follows-mouse, it a negative. To get focus-follows-mouse in Aqua, type the following in your Terminal window: defaults write com.apple.Terminal FocusFollowsMouse -string YES and then logout and log in again or quit and restart Terminal. I had thought I originally got that useful hint from your own fabulous PX on OSX pages but clearly not. best wishes Pete Artymiuk On 20 Jan 2008, at 15:39, William Scott wrote: Hi David: david lawson (JIC) wrote: Dear All, Sorry for the slightly off-topic subject. We have recently bought a few iMacs for crystallography. I'm not keen on the supplied mighty mouse May I have them? so I have switched to using a microsoft 3-button wheel mouse. I would like to configure it so that it behaves as it would with other unix systems such as RH Linux. You managed to use Microsoft, behaved and Linux (albeit RH) all in one sentence without a hint of irony. i.e. (1) double-clicking with LH button on a file name selects ALL of the file name, not just up to the first full stop. Although your choice of Microsoft products shows dedication to a company with a firm reputation for placing the customizability needs of its customers ahead of its own desire to make profits, the first thing to realize is that you should never ever ever install their drivers. Ever. So if you did, take them out, now, and reboot. I'll wait. It is still early Sunday morning here. (2) clicking the scroll wheel pastes the selected text AND it can be done multiple times without re-selecting. When you've gotten rid of the drivers, this should now work. In Apple's Terminal program (as of 10.5) and iTerm (as of 1215), you just set the preference to do middle-button-paste and left-button select, and Blair's your uncle. Unfortunately, in pretty much every other application I can think of on OS X, this, sadly, does not work, and there is nothing Steve Gates will let you do about it. (2) I would like these functions to work in terminal windows, the ccp4i gui and web pages (and probably a few other things I haven't thought of yet!) AND be able to transfer the selected text between applications. I'd like to be at my ideal high-school weight, be paid more than a postdoc, and, well ... Getting the OS X gui to play nice with X11 is sometimes challenging. With the exception of Terminal and iTerm, you have to explicitly put stuff in the copy/paste buffer (command-C) before it is in the system clipboard. Then you can paste to X11 programs with a middle-button click, but this only works if you uninstalled that viral driver. Going from X11 to aqua programs requires selecting the text in the usual X11 manner but explicitly issuing the paste command (command- p). If you are using KDE X11 applications, you are really in for headaches. To get whole-string selection in iTerm or Terminal, there is a preference setting that allows you to input which characters you want to have considered parts of a word for click-to-select purposes. Unfortunately, pretty much every other application lacks this customizability, and I know of no system-wide preference setting that would enable you to do this globally. Aqua simply behaves by slightly different rules. Although I am a slobbering OS X fan, this lack of customizability to me, as well as a lack of focus-follows-mouse, it a negative. If you really need the canonical linux behavior, you can install gnome, xfce4, KDE, enlightenment, or any number of other window managers via fink. I've found KDE buggy and the XFCE4 is way out of date. Gnome is probably the best bet, and there is a major effort now to bring it completely up to date in fink. I have installed the microsoft intellipoint drivers that seem to give more control over configuring the various buttons through system preferences, but I still can't get what I want. Therein lies the problem, I am afraid. OS X will behave better using the default settings. It may be possible to tinker around with the driver, including separate settings in X11, to recover canonical behavior, but for purposes of sanity, uninstall them first, get everything working as best as possible, verify middle-button-paste works in X11, verify X11 coot and pymol do the right thing, and then if you need additional functionality, reinstall the drivers, verify things like coot and pymol still use the middle button correctly, or adjust until they do, and only then try customizing. Best of luck! Bill Any help would be much appreciated. Many thanks Dave Lawson --- Dr. David M. Lawson Biological Chemistry Dept., John Innes Centre, Norwich, NR4 7UH, UK. Tel: +44-(0)1603-450725 Fax: +44-(0)1603-450018 Email: [EMAIL PROTECTED] Web:
Re: [ccp4bb] Why there are difference density when occupancy is 1.00
Hard to say without more information. Have you refined the B factors for these residues? Most building software gives some arbitrary b value, which must then be refined. (In fact after any rebuilding activity you need to do a few cycles of refinement before looking at the maps again) Eleanor Sun Tang wrote: Hello Everyone, When I refined a structure, I found strong difference density Fo-Fc at 3 sigma contour for the for five residues which already have occupancy of 1. The density is continuous and so strong as if I did not put the residues there. Why was that? Can I put greater than 1 occupancy for those residues? Thank you very much for your opinions and suggestions! Best wishes, Sun Tang - Never miss a thing. Make Yahoo your homepage.
[ccp4bb] Question about freeR tag
Hi, All Could any tell me how CCP4 handle free R flag? I know It is important to select the same** FreeR reflections if I move to next step of refinement. But everytime I start from fresh, the freeR Flag remains unchanged. The Rwork and Rfree of my models are fine (20.7% and 22.9%). I thought Rfree was randomly generated. I asked more experienced people in the lab and neighbor labs, and didn't get a straight answer. Did I do something wrong? Thanks and sorry to bother others. Zheng (Joe)
Re: [ccp4bb] microsoft 3-button wheel mouse with OS X 10.5
Yes, thanks, that does it for the Terminal.app, but not for any of the rest. It would be great to have such a feature globally. mb1pja wrote: Dear Bill William Scott wrote Aqua simply behaves by slightly different rules. Although I am a slobbering OS X fan, this lack of customizability to me, as well as a lack of focus-follows-mouse, it a negative. To get focus-follows-mouse in Aqua, type the following in your Terminal window: defaults write com.apple.Terminal FocusFollowsMouse -string YES and then logout and log in again or quit and restart Terminal. I had thought I originally got that useful hint from your own fabulous PX on OSX pages but clearly not. best wishes Pete Artymiuk On 20 Jan 2008, at 15:39, William Scott wrote: Hi David: david lawson (JIC) wrote: Dear All, Sorry for the slightly off-topic subject. We have recently bought a few iMacs for crystallography. I'm not keen on the supplied mighty mouse May I have them? so I have switched to using a microsoft 3-button wheel mouse. I would like to configure it so that it behaves as it would with other unix systems such as RH Linux. You managed to use Microsoft, behaved and Linux (albeit RH) all in one sentence without a hint of irony. i.e. (1) double-clicking with LH button on a file name selects ALL of the file name, not just up to the first full stop. Although your choice of Microsoft products shows dedication to a company with a firm reputation for placing the customizability needs of its customers ahead of its own desire to make profits, the first thing to realize is that you should never ever ever install their drivers. Ever. So if you did, take them out, now, and reboot. I'll wait. It is still early Sunday morning here. (2) clicking the scroll wheel pastes the selected text AND it can be done multiple times without re-selecting. When you've gotten rid of the drivers, this should now work. In Apple's Terminal program (as of 10.5) and iTerm (as of 1215), you just set the preference to do middle-button-paste and left-button select, and Blair's your uncle. Unfortunately, in pretty much every other application I can think of on OS X, this, sadly, does not work, and there is nothing Steve Gates will let you do about it. (2) I would like these functions to work in terminal windows, the ccp4i gui and web pages (and probably a few other things I haven't thought of yet!) AND be able to transfer the selected text between applications. I'd like to be at my ideal high-school weight, be paid more than a postdoc, and, well ... Getting the OS X gui to play nice with X11 is sometimes challenging. With the exception of Terminal and iTerm, you have to explicitly put stuff in the copy/paste buffer (command-C) before it is in the system clipboard. Then you can paste to X11 programs with a middle-button click, but this only works if you uninstalled that viral driver. Going from X11 to aqua programs requires selecting the text in the usual X11 manner but explicitly issuing the paste command (command- p). If you are using KDE X11 applications, you are really in for headaches. To get whole-string selection in iTerm or Terminal, there is a preference setting that allows you to input which characters you want to have considered parts of a word for click-to-select purposes. Unfortunately, pretty much every other application lacks this customizability, and I know of no system-wide preference setting that would enable you to do this globally. Aqua simply behaves by slightly different rules. Although I am a slobbering OS X fan, this lack of customizability to me, as well as a lack of focus-follows-mouse, it a negative. If you really need the canonical linux behavior, you can install gnome, xfce4, KDE, enlightenment, or any number of other window managers via fink. I've found KDE buggy and the XFCE4 is way out of date. Gnome is probably the best bet, and there is a major effort now to bring it completely up to date in fink. I have installed the microsoft intellipoint drivers that seem to give more control over configuring the various buttons through system preferences, but I still can't get what I want. Therein lies the problem, I am afraid. OS X will behave better using the default settings. It may be possible to tinker around with the driver, including separate settings in X11, to recover canonical behavior, but for purposes of sanity, uninstall them first, get everything working as best as possible, verify middle-button-paste works in X11, verify X11 coot and pymol do the right thing, and then if you need additional functionality, reinstall the drivers, verify things like coot and pymol still use the middle button correctly, or adjust until they do, and only then try customizing. Best of luck! Bill Any help would be much appreciated. Many thanks Dave Lawson --- Dr. David M.
[ccp4bb] problem on protein precipitation
Hi ccp4ers, Sorry for this out-topic question: Recently we have a membrane protein expressed, after solubilized with detergent and purified from IMAC, the protein looks beautiful in SEC. However, it completely precipitates after the 2-3 days storage in 4 degree. We supplement 2 mM DTT in the new elute from IMAC, the protein looks happy during weeks at 4 degree. However, it starts to form an invisible aggregate (verified from SEC) during the protein concentration by Centricon. I know this is not uncommon problem for both soluble and membrane proteins and wonder if anyone has any tip and experience to overcome this problem. The protein pI is 8.6, buffer used is pH 7.6. Glycerol is always present during the purification. We do have high salt (500mM) in the buffer. Thank you for you input in advance, Lei
Re: [ccp4bb] Question about freeR tag
Sorry that I didn't explain the situation clearly. I used only one output.sca file from HLK2000. I ran the scalepack2mtz (in CCP4i, data reduction, import merged data) several times, on both linux Fedora and window XP, the same computer though. For the next step refinement, I mean add H2O, ion, ligands, double check certain sidechains In those refmac runs, I always use the same mtz file, which I generated from the beginning. I am as surprised as you are the Rfree flags are identical from those different first runs. Thanks On Jan 23, 2008 9:50 AM, Zheng Zhou [EMAIL PROTECTED] wrote: Hi, All Could any tell me how CCP4 handle free R flag? I know It is important to select the same** FreeR reflections if I move to next step of refinement. But everytime I start from fresh, the freeR Flag remains unchanged. The Rwork and Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree was randomly generated. I asked more experienced people in the lab and neighbor labs, and didn't get a straight answer. Did I do something wrong? Thanks and sorry to bother others. Zheng (Joe) I cant understand this. Do you mean you have multiple data sets and they all generate the same FreeRs? If you have EXACTLY the same reflection list, and generate FreeR flags on the same machine I guess they would be the same.. but it would be surprising Or if you are using the same file which already has FreeR flags then they wont change of course - the default is always to use the reflections flagged with FreeR = 0. (Thats as it should be..) By the by - your agreement between R and FreeR seems unusually low unless you have very high resolution data or a great deal of non-crystallographic symmetry. Eleanor Zheng - I am not sure what you are doing, but as long as you work with one dataset only, you generate the free reflections at the very beginning when you import your data into CCP4. Then use the resulting mtz file for all following refinement steps. There are situations where one will have to deviate from this scheme, but those are rare. If you feel you have such a case, then tell us more about it. Hope that helps. Best - MM Joe, can you explain a bit clearer your problem? what do you mean the 'next step of refinement'? are you just doing another round of refinement? what program are you using, REFMAC5? also, R/Rfree of 20.7/22.9 is pretty good, depending on resolution. yes, you are right, Rfree flags are generated randomly and can be anywhere from 5-10% of your reflections (your choice). then, once the Rfree flagged reflections are selected, they will not change (nor be refined) throughout the rest of your structure determination. they should have their own column in your *.mtz file called RfreeFlag or something like this (in CCP4 that is). other programs handle this relatively the same, but may use various naming conventions. one caveat is that CCP4 flags reflections using 0 by default (meaning 5% of your reflections are flagged with 0 and are the Rfree set). Other programs such as CNS and PHENIX use 1 by default, so be careful when switching back and forth. You can tell either program which to use for Rfree set, but needs to be set, since it is not default. again, not exactly sure what the problem is, but i hope some of this helps. feel free to email with further questions if needed. best of luck! cheers, nick
Re: [ccp4bb] problem on protein precipitation
Hi Lei, Try this: 50-100 mM Arginine in your buffers. Or Glutamic Acid. Or both. -- Lisa A. Nagy, Ph.D. University of Alabama-Birmingham [EMAIL PROTECTED] From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Zheng, Lei Sent: Wednesday, January 23, 2008 10:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] problem on protein precipitation Hi ccp4ers, Sorry for this out-topic question: Recently we have a membrane protein expressed, after solubilized with detergent and purified from IMAC, the protein looks beautiful in SEC. However, it completely precipitates after the 2-3 days storage in 4 degree. We supplement 2 mM DTT in the new elute from IMAC, the protein looks happy during weeks at 4 degree. However, it starts to form an invisible aggregate (verified from SEC) during the protein concentration by Centricon. I know this is not uncommon problem for both soluble and membrane proteins and wonder if anyone has any tip and experience to overcome this problem. The protein pI is 8.6, buffer used is pH 7.6. Glycerol is always present during the purification. We do have high salt (500mM) in the buffer. Thank you for you input in advance, Lei
Re: [ccp4bb] Question about freeR tag
Zheng Zhou wrote: Hi, All Could any tell me how CCP4 handle free R flag? I know It is important to select the same** FreeR reflections if I move to next step of refinement. But everytime I start from fresh, the freeR Flag remains unchanged. The Rwork and Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree was randomly generated. I asked more experienced people in the lab and neighbor labs, and didn't get a straight answer. Did I do something wrong? Thanks and sorry to bother others. Zheng (Joe) You have two options for setting the FreeR flag in CCP4i. You can do it at the time of scaling in SCALA by ticking the button Ensure unique data add FreeR column for x.xx fraction of data. 5% of the data is the default, which may be excessive depending on the number of total reflections you have. If you did not set the FreeR flag during scaling, you can do it later using the Convert to MTZ Standardise task under Reflection Utilities in the GUI. Tick Create a full unique set of reflections and generate FreeR data and select your FreeR fraction in the box near the bottom of the task window. Once the FreeR flag is set it should not change, and you should not generate a new set of FreeR flags again during the refinement. The idea is to set aside some data early on that is never used to refine the model so that it is as unbiased as possible in evaluating your refinement statistics and guarding against model bias. When refining with Refmac5, tick the Exclude data with freeR label FreeR_flag with value of 0 to ignore the FreeR data during refinement. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] Why there are difference density when occupancy is 1.00
Hi James, I did check teh B-factors and they are similar to the flanking regions (about 40). The difference density appeared at the later stage of refinement (TLS and restrained in CCP4i). What do you think and how to do it? Best, Sun Tang James Irving [EMAIL PROTECTED] wrote: Hi Sun, I suggest checking that the b-factors for those residues aren't unexpectedly high compared to those in the flankinh regions. Cheers, James On 1/23/08, Sun Tang wrote: Hello Everyone, When I refined a structure, I found strong difference density Fo-Fc at 3 sigma contour for the for five residues which already have occupancy of 1. The density is continuous and so strong as if I did not put the residues there. Why was that? Can I put greater than 1 occupancy for those residues? Thank you very much for your opinions and suggestions! Best wishes, Sun Tang - Never miss a thing. Make Yahoo your homepage. -- Sent from Gmail for mobile | mobile.google.com Dr. James Irving NHMRC C.J. Martin Fellow School of Biomedical Sciences Building 13D Monash University Wellington Road Melbourne 3800 Australia - Never miss a thing. Make Yahoo your homepage.
Re: [ccp4bb] Question about freeR tag
FREEFLAG (the program which is used to generate the test set) description says when describing the keyword SEED: By default, for a given job on a given machine, the random number generator produces the same list of random free-R flags each time the job is run. Since you would generally only produce one list of free-R flags for each project, this is not usually a problem. However, if you specify the keyword SEED, then the random number generator is seeded with the current time, and will produce a different list of free-R flags each time the job is run. So what you see is normal. Zheng Zhou wrote: Sorry that I didn't explain the situation clearly. I used only one output.sca file from HLK2000. I ran the scalepack2mtz (in CCP4i, data reduction, import merged data) several times, on both linux Fedora and window XP, the same computer though. For the next step refinement, I mean add H2O, ion, ligands, double check certain sidechains In those refmac runs, I always use the same mtz file, which I generated from the beginning. I am as surprised as you are the Rfree flags are identical from those different first runs. Thanks On Jan 23, 2008 9:50 AM, Zheng Zhou [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] wrote: Hi, All Could any tell me how CCP4 handle free R flag? I know It is important to select the same FreeR reflections if I move to next step of refinement. But everytime I start from fresh, the freeR Flag remains unchanged. The Rwork and Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree was randomly generated. I asked more experienced people in the lab and neighbor labs, and didn't get a straight answer. Did I do something wrong? Thanks and sorry to bother others. Zheng (Joe) I cant understand this. Do you mean you have multiple data sets and they all generate the same FreeRs? If you have EXACTLY the same reflection list, and generate FreeR flags on the same machine I guess they would be the same.. but it would be surprising Or if you are using the same file which already has FreeR flags then they wont change of course - the default is always to use the reflections flagged with FreeR = 0. (Thats as it should be..) By the by - your agreement between R and FreeR seems unusually low unless you have very high resolution data or a great deal of non-crystallographic symmetry. Eleanor Zheng - I am not sure what you are doing, but as long as you work with one dataset only, you generate the free reflections at the very beginning when you import your data into CCP4. Then use the resulting mtz file for all following refinement steps. There are situations where one will have to deviate from this scheme, but those are rare. If you feel you have such a case, then tell us more about it. Hope that helps. Best - MM Joe, can you explain a bit clearer your problem? what do you mean the 'next step of refinement'? are you just doing another round of refinement? what program are you using, REFMAC5? also, R/Rfree of 20.7/22.9 is pretty good, depending on resolution. yes, you are right, Rfree flags are generated randomly and can be anywhere from 5-10% of your reflections (your choice). then, once the Rfree flagged reflections are selected, they will not change (nor be refined) throughout the rest of your structure determination. they should have their own column in your *.mtz file called RfreeFlag or something like this (in CCP4 that is). other programs handle this relatively the same, but may use various naming conventions. one caveat is that CCP4 flags reflections using 0 by default (meaning 5% of your reflections are flagged with 0 and are the Rfree set). Other programs such as CNS and PHENIX use 1 by default, so be careful when switching back and forth. You can tell either program which to use for Rfree set, but needs to be set, since it is not default. again, not exactly sure what the problem is, but i hope some of this helps. feel free to email with further questions if needed. best of luck! cheers, nick -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Question about freeR tag
Thank you all for fast replying. The reason that I am trying to use a different set of FreeR flag from the very beginning of the refinement is that for some data set, my colleague's CNS refinement gave converged Rfree and R work, 3% difference. However both my CNS and CCP4 refinement gave a difference of about 7%. I was trying to use the same parameter settings as my colleague in simulated annealing. His program is from his previous lab and all the input files for CNS are modified as a batch file. But mine are just individual files downloaded from CNS website. Among those things I tried, I thought about looking at the true input data for computing Rfree and Rwork. It appeared to me the FreeR flags in my First runs are never randomized. Thanks for your reply, I guess I could use a different flag number other than 0 (I know I am not supposed to change this in the middle of a refinement). I even tried to use my colleague's cross-validation file .cv for CNS. It still diverge in my runs. Anyone met similar problem before, where CNS and CCP4 give a quite different R factors? Thanks for your insight. Zheng (Joe) On Jan 23, 2008 9:50 AM, Zheng Zhou [EMAIL PROTECTED] wrote: Hi, All Could any tell me how CCP4 handle free R flag? I know It is important to select the same** FreeR reflections if I move to next step of refinement. But everytime I start from fresh, the freeR Flag remains unchanged. The Rwork and Rfree of my models are fine ( 20.7% and 22.9%). I thought Rfree was randomly generated. I asked more experienced people in the lab and neighbor labs, and didn't get a straight answer. Did I do something wrong? Thanks and sorry to bother others. Zheng (Joe)
[ccp4bb] meeting suggestions
Dear All, I have a probably quite controversial question for the crystallographic community (and there may be a strong personal bias too...). Our group would like to select 4 meetings this year that would really be focused towards our line of work (protein crystallography in collaboration with drug design) and that are US-based. We have started to prepare a list but may have missed some and/or need to select from that list. So, if you were in our position, what would your top 4 be? All suggestions welcome... Thank you very much for your time and help, Ingrid Mechin Ingrid Mechin Research Investigator Chemical and Analytical Sciences sanofi-aventis mailstop N-103A route 202-206 Bridgewater NJ 08807 USA tel: + 1 908 231 3348 fax: + 1 908 231 3576 e-mail:[EMAIL PROTECTED]
[ccp4bb] GPCR Structural Biology Postdoctoral Position Openings
GPCR Structural Biology Postdoctoral Position Openings We have several openings for postdoctoral fellows in the area of GPCR structural biology in the Kuhn-Stevens Laboratory at The Scripps Research Institute. With the recent structure determination of the human beta2 adrenergic receptor (Cherezov et al; Rosenbaum et al, Science 2007), we are now interested in studying other G-protein coupled receptors as well as focus on the mechanistic details of a single G-protein signal transduction system. The Kuhn-Stevens laboratory is well equipped with cutting edge technologies in the areas of membrane protein expression, stabilization, and crystallization. Applicants interested in the biology, biochemistry and/or structural biology of this family of membrane proteins are encouraged to apply. Interested applicants should send their CV, statement of research interest, and 3 letters of reference to [EMAIL PROTECTED]
