Re: [ccp4bb] question about the zinc binding protein
Hi, I'm working with a possible zinc binding protein. Saw your post and was wondering, what is the proper pH range for zinc binding? Thanks, Peter
Re: [ccp4bb] protein degradation during concentration for crystallization trials
Hi, I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein? Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to nearly water may not be great thing to do. Can your protein concentrate in your purification buffer to about .1-.2mM? If it can, I highly suggest trying to use the dialysis buttons from Hampton and screen a variety of different pHs, salts and additives to see what your protein can tolerate, before committing an entire purification prep to concentration. I've also noticed if you're using the centricon concentrators, make sure you take it out every few minutes and pipette it the solution a bit. These concentrators tend to create a concentration gradient, making the local concentration of protein very high in bottom of the concentrator, often times resulting in ppt. Hope it helps
Re: [ccp4bb] programs to check the structure of DNA
Ciao Alessandra, A couple of pointers: http://gbio-pbil.ibcp.fr/Curves_plus/Curves+.html http://rutchem.rutgers.edu/~xiangjun/3DNA/ HTH, Luca Luca Jovine, Ph.D. Group Leader EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.6083-301 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org On Apr 9, 2010, at 10:25 AM, Alessandra Pesce wrote: Dear All, I am looking for available programs and/or websites able to check the structure of DNA in DNA-protein complexes. I need to evaluate the DNA bending, its backbone distortion, the alteration of the base pairing, and its interaction with the protein. The aim is to compare in easy and quick way different conformation of DNA in different DNA-protein complexes. Can anybody help me? Thanks in advance. Cheers Alessandra * Alessandra Pesce, PhD Department of Physics University of Genova Via Dodecaneso 33 16146 Genova, Italy Tel.(DIFI) ++39 010 353 6243 | 6309 e-mail pe...@ge.infm.it *
[ccp4bb] freezing crystals grown in isopropanol condition
Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris
[ccp4bb] Does the substrate has access to the active site?
Dear Bulletin Board, I am trying to soak substrate into crystals of an enzyme, but so far I can't see the substrate in the structure. Does anyone knows a program to ensure that the entrance to the central cavity is accessibly? I mean based on the whole crystal. I already checked the crystal packing manually and it seems that the way is free more or less, but I find it hard to interpret. Thanks in advance, P.
Re: [ccp4bb] freezing crystals grown in isopropanol condition
Dear Chris, I recall from many years ago that we had a screening hit in high isopropanol, which started boiling violently when we opened the well. We solved the problem by using another precipitant but I would also be interested in tricks how to handle high isopropanol. Best regards, Herman From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Chris Meier Sent: Friday, April 09, 2010 10:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] freezing crystals grown in isopropanol condition Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris
Re: [ccp4bb] Does the substrate has access to the active site?
Paul, couple things come to mind: -make sure your substrate is actually a binder and not an (unspecific) inhibitor (ITC, Thermofluor, Biacore) -soak longer and at higher concentration. (3mM concentration from 100mM stock in DMSO for a week or more) -Is there evidence that your protein needs to undergo substantial rearrangement prohibited by the crystal packing? Then you may be out of luck with soaking, try co-crystallization. The folks from GSK had a nice paper about some useful techniques for ligand incorporation probably a worthwhile read: Acta Cryst. (2007). D63, 72-79 (doi:10.1107/S0907444906047020) HTH Carsten -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Paul Lindblom Sent: Friday, April 09, 2010 5:15 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Does the substrate has access to the active site? Dear Bulletin Board, I am trying to soak substrate into crystals of an enzyme, but so far I can't see the substrate in the structure. Does anyone knows a program to ensure that the entrance to the central cavity is accessibly? I mean based on the whole crystal. I already checked the crystal packing manually and it seems that the way is free more or less, but I find it hard to interpret. Thanks in advance, P.
Re: [ccp4bb] Does the substrate has access to the active site?
It is also possible that mother liquor prevents binding (although often in such cases you would see some precipitant component in the active site. I would generally bet on need for conformational change. And you expect to see the product complex, right? Ed. On Fri, 2010-04-09 at 11:15 +0200, Paul Lindblom wrote: Dear Bulletin Board, I am trying to soak substrate into crystals of an enzyme, but so far I can't see the substrate in the structure. Does anyone knows a program to ensure that the entrance to the central cavity is accessibly? I mean based on the whole crystal. I already checked the crystal packing manually and it seems that the way is free more or less, but I find it hard to interpret. Thanks in advance, P. -- 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] Follow up to TLS, NCS and refinement
On Thu, 2010-04-08 at 23:26 +0100, Daniel Bonsor wrote: both the Rfactor and Rfree get stuck at 30% and 36% according to http://xray.bmc.uu.se/gerard/supmat/rfree2000/plotter.html these are higher than expected. With that said, R/Rfree should not be a fetish, and your model may be fine (i.e. as good as it could be given the data quality). Definitely re-check if you have the right space group. This may be painful, as dropping some symmetry will only increase the number of molecules in the asu, and you already have 8. Sometimes, however, R-factors stay higher-than-expected no matter what you do. It may be driven by noise in the low resolution domain, and you may consider re-processing the data more carefully. Or, if you have more crystals, just collect more data and/or try different cryoprotection. Cheers, Ed. -- 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] freezing crystals grown in isopropanol condition
Yes, isopropanol is a cryoprotectant, and a relatively good one. So are the other alcohols. It was even popular in the olden days when we would typically set up drops that were 5-10 microliters in volume (each!). These take a while (minutes) to evaporate, giving you enough working time to mount the crystal before the alcohol concentration changed too much. Modern nanoliter-scale drops have largely made alcohol additives impractical, which is a shame. A potentially general way to deal with evaporating drops is to bathe the work area in a stream of air or nitrogen that has been pre-saturated with the reservoir solution. That is, run the gas line in and out of a jar of say about 50-100 mL of replicated reservoir solution (bubbling the gas through the solution in the jar) and then route the end of the hose to under your dissecting microscope and point it at your crystallization well just before you crack it open. This should give you a nice, long working time, and similar devices have already been reported in the literature: http://dx.doi.org/10.1107/S0021889801020702 That, or you can try to just work really quickly! -James Holton MAD Scientist Chris Meier wrote: Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris
Re: [ccp4bb] programs to check the structure of DNA
I know two programs; 3DNA by Lu and curves by Lavery and Sklenar. 3DNA is easier to use, it can also make input for the Curves. Maia Alessandra Pesce wrote: Dear All, I am looking for available programs and/or websites able to check the structure of DNA in DNA-protein complexes. I need to evaluate the DNA bending, its backbone distortion, the alteration of the base pairing, and its interaction with the protein. The aim is to compare in easy and quick way different conformation of DNA in different DNA-protein complexes. Can anybody help me? Thanks in advance. Cheers Alessandra * Alessandra Pesce, PhD Department of Physics University of Genova Via Dodecaneso 33 16146 Genova, Italy Tel.(DIFI) ++39 010 353 6243 | 6309 e-mail pe...@ge.infm.it mailto:pe...@ge.infm.it *
Re: [ccp4bb] freezing crystals grown in isopropanol condition
I have wondered if placing a layer of oil over the drop would help solve the problem of the crystals moving around. Haven't tried it, but don't people harvest from a microbatch tray by dragging the loop and crystal through oil? Nat On Fri, Apr 9, 2010 at 11:21 AM, James Holton jmhol...@lbl.gov wrote: Yes, isopropanol is a cryoprotectant, and a relatively good one. So are the other alcohols. It was even popular in the olden days when we would typically set up drops that were 5-10 microliters in volume (each!). These take a while (minutes) to evaporate, giving you enough working time to mount the crystal before the alcohol concentration changed too much. Modern nanoliter-scale drops have largely made alcohol additives impractical, which is a shame. A potentially general way to deal with evaporating drops is to bathe the work area in a stream of air or nitrogen that has been pre-saturated with the reservoir solution. That is, run the gas line in and out of a jar of say about 50-100 mL of replicated reservoir solution (bubbling the gas through the solution in the jar) and then route the end of the hose to under your dissecting microscope and point it at your crystallization well just before you crack it open. This should give you a nice, long working time, and similar devices have already been reported in the literature: http://dx.doi.org/10.1107/S0021889801020702 That, or you can try to just work really quickly! -James Holton MAD Scientist Chris Meier wrote: Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris
Re: [ccp4bb] freezing crystals grown in isopropanol condition
Yes, oil is great, but you have to be careful to choose an oil in which the alcohol is not soluble, or the oil will suck it out of your drop, (just like air). This is particularly annoying with detergents, which are almost all soluble in oil. I've always thought that maybe some synthetic motor oils (which your auto mechanic will tell you are immiscible with petroleum-based oils) might be a good thing to try with membrane proteins. It is a common trick, however, to pre-saturate the oil by vortexing it with an excess of the reservoir solution before applying it to the drop. Obviously, however, this trick can get expensive when working with detergents... -James Holton MAD Scientist Nathaniel Clark wrote: I have wondered if placing a layer of oil over the drop would help solve the problem of the crystals moving around. Haven't tried it, but don't people harvest from a microbatch tray by dragging the loop and crystal through oil? Nat On Fri, Apr 9, 2010 at 11:21 AM, James Holton jmhol...@lbl.gov wrote: Yes, isopropanol is a cryoprotectant, and a relatively good one. So are the other alcohols. It was even popular in the olden days when we would typically set up drops that were 5-10 microliters in volume (each!). These take a while (minutes) to evaporate, giving you enough working time to mount the crystal before the alcohol concentration changed too much. Modern nanoliter-scale drops have largely made alcohol additives impractical, which is a shame. A potentially general way to deal with evaporating drops is to bathe the work area in a stream of air or nitrogen that has been pre-saturated with the reservoir solution. That is, run the gas line in and out of a jar of say about 50-100 mL of replicated reservoir solution (bubbling the gas through the solution in the jar) and then route the end of the hose to under your dissecting microscope and point it at your crystallization well just before you crack it open. This should give you a nice, long working time, and similar devices have already been reported in the literature: http://dx.doi.org/10.1107/S0021889801020702 That, or you can try to just work really quickly! -James Holton MAD Scientist Chris Meier wrote: Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris
Re: [ccp4bb] freezing crystals grown in isopropanol condition
Nat, A few years ago I had K channel crystals that formed under similar conditions. I found that using MPD as a cryo-protectant worked best. As for the evaporation issue, I had a little extra time as I was performing the mounting in a cold room. Scott Pegan S, Arrabit C, Zhou W, Kwiatkowski W, Collins A, Slesinger PA, Choe S. Nat Neurosci. 2005 Mar;8(3):279-87. Epub 2005 Feb 20. On Fri, Apr 9, 2010 at 9:59 AM, James Holton jmhol...@lbl.gov wrote: Yes, oil is great, but you have to be careful to choose an oil in which the alcohol is not soluble, or the oil will suck it out of your drop, (just like air). This is particularly annoying with detergents, which are almost all soluble in oil. I've always thought that maybe some synthetic motor oils (which your auto mechanic will tell you are immiscible with petroleum-based oils) might be a good thing to try with membrane proteins. It is a common trick, however, to pre-saturate the oil by vortexing it with an excess of the reservoir solution before applying it to the drop. Obviously, however, this trick can get expensive when working with detergents... -James Holton MAD Scientist Nathaniel Clark wrote: I have wondered if placing a layer of oil over the drop would help solve the problem of the crystals moving around. Haven't tried it, but don't people harvest from a microbatch tray by dragging the loop and crystal through oil? Nat On Fri, Apr 9, 2010 at 11:21 AM, James Holton jmhol...@lbl.gov wrote: Yes, isopropanol is a cryoprotectant, and a relatively good one. So are the other alcohols. It was even popular in the olden days when we would typically set up drops that were 5-10 microliters in volume (each!). These take a while (minutes) to evaporate, giving you enough working time to mount the crystal before the alcohol concentration changed too much. Modern nanoliter-scale drops have largely made alcohol additives impractical, which is a shame. A potentially general way to deal with evaporating drops is to bathe the work area in a stream of air or nitrogen that has been pre-saturated with the reservoir solution. That is, run the gas line in and out of a jar of say about 50-100 mL of replicated reservoir solution (bubbling the gas through the solution in the jar) and then route the end of the hose to under your dissecting microscope and point it at your crystallization well just before you crack it open. This should give you a nice, long working time, and similar devices have already been reported in the literature: http://dx.doi.org/10.1107/S0021889801020702 That, or you can try to just work really quickly! -James Holton MAD Scientist Chris Meier wrote: Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry Biochemistry University of Denver
Re: [ccp4bb] programs to check the structure of DNA
Hi Alessandra, Here is a list of nucleic acid and protein-nucleic acid related tools (including the ones mentioned by Luca and Maia: 3DNA http://rutchem.rutgers.edu/~xiangjun/3DNA/ http://w3dna.rutgers.edu/ curves http://gbio-pbil.ibcp.fr/Curves_plus/Curves+.html nucplot http://www.biochem.ucl.ac.uk/bsm/nucplot.html protein-nucleic acid interaction server http://www.biochem.ucl.ac.uk/bsm/DNA/server/ freehelix ftp://ndbserver.rutgers.edu/NDB/programs/freehelix98/README In addition, general analysis tools might be useful as well: difference distance matrix http://www.roselab.jhu.edu/ddmp/ moleman2 (distance commands and others) http://xray.bmc.uu.se/usf/xutil.html lsqman (plots; superpositions of nucleic acids possible, at ph or define your own atom type) http://xray.bmc.uu.se/usf/dejavu.html ... Best regards, christian Alessandra Pesce wrote: Dear All, I am looking for available programs and/or websites able to check the structure of DNA in DNA-protein complexes. I need to evaluate the DNA bending, its backbone distortion, the alteration of the base pairing, and its interaction with the protein. The aim is to compare in easy and quick way different conformation of DNA in different DNA-protein complexes. Can anybody help me? Thanks in advance. Cheers Alessandra * Alessandra Pesce, PhD Department of Physics University of Genova Via Dodecaneso 33 16146 Genova, Italy Tel.(DIFI) ++39 010 353 6243 | 6309 e-mail pe...@ge.infm.it mailto:pe...@ge.infm.it * -- ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
[ccp4bb] ALS or Uni Pucks
Does anyone know the status of Crystal Positioning? They are not responding to email or phone calls. Are they the only alternative to the late Peter Boyd? Deena Deena Abells Oren, PhD Manager, Structural Biology Resource Center Rockefeller University 1230 York Avenue, Box 295 New York, NY 10065-6399 phone: 212- 327-7429 fax: 212-327-7389
[ccp4bb] fink update
Hi, Sorry for a non-ccp4 question. I hope there is someone who can solve the problem. I was trying to update fink and I got the following error. rajadey$ fink update-all Password: Information about packages read in 2 seconds. fink needs help picking an alternative to satisfy a virtual dependency. The candidates: (1)db48-aes: Berkeley DB embedded database - crypto (2)db48: Berkeley DB embedded database - non crypto Pick one: [1] Can't resolve dependency xcode (= 3.1.2) for package gcc44-4.4.2-1000 (no matching packages/versions found) Exiting with failure. Should I select the non crypto candidate? Thanking you in advance... Raja
[ccp4bb] R-free ratio, effect of ncs restraints?
Has anyone looked theoretically at how ncs-restraints affect the expected Rfree/R ratio? Tickle et al., Acta Cryst. (1998). D54, 547-557 concluded Rfree/R = sqrt(Nobs/(Nobs-Nparam)) . He suggested that, with restrained refinement of coordinates plus individual isotropic B-factors, the effective number of parameters per atom is two. If we add strong N-fold NCS restraints on coordinates and B-factor, does that effectively reduce the number of parameters by a factor of N? Giving 2/N for parameters per atom? I'm curious how much of the drop in the r-free ratio observed on enforcing NCS is due to the reduction in the effective number of parameters, and how much is due to linking reflections in the free set with the working set. Given an expression to predict the effect of reducing number of parameters, seeing how much of the actual drop in Rfree/R it accounts for would let us see how severe the linkage problem is. Ed
Re: [ccp4bb] fink update
Hi Raja,I assume you are running OS X?I had this same problem since I was running xcode v3.0. The new compiler (the one its complaining about) should be packaged with the xcode on the snow leopard disc, which I believe is the most recent version. I could be wrong, since I haven't yet fixed my own problem, but I think the error you see is simply from an outdated xcode. Best regards,Jason-CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote: -To: CCP4BB@JISCMAIL.AC.UKFrom: Raja Dey deyra...@yahoo.co.inSent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UKDate: 04/09/2010 02:21PMSubject: [ccp4bb] fink updateHi, Sorry for a non-ccp4 question. I hope there is someone who can solve the problem. I was trying to update fink and I got the following error.rajadey$ fink update-allPassword:Information about packages read in 2 seconds.fink needs help picking an alternative to satisfy a virtual dependency. Thecandidates:(1) db48-aes: Berkeley DB embedded database - crypto(2) db48: Berkeley DB embedded database - non cryptoPick one: [1] Can't resolve dependency "xcode (= 3.1.2)" for package "gcc44-4.4.2-1000" (nomatching packages/versions found)Exiting with failure.Should I select the non crypto candidate?Thanking you in advance...Raja
Re: [ccp4bb] ALS or Uni Pucks
We have drawings available for both pucks and tools, freely available for anybody to use: http://bcsb.als.lbl.gov/wiki/index.php/Automounter#Technical_drawings_and_CAD_models_for_the_pucks_and_automounter_tools At the moment, crystal positioning is the only supplier possible (they will have the ALS puck as well AFAIK). If there is demonstrable demand from the user community for these items, other vendors might have interest as well. Systems like the Actor, BAM and Irelec CATS systems can/should handle these pucks. HTH Peter 2010/4/9 Deena Oren do...@mail.rockefeller.edu: Does anyone know the status of Crystal Positioning? They are not responding to email or phone calls. Are they the only alternative to the late Peter Boyd? Deena Deena Abells Oren, PhD Manager, Structural Biology Resource Center Rockefeller University 1230 York Avenue, Box 295 New York, NY 10065-6399 phone: 212- 327-7429 fax: 212-327-7389 -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] R-free ratio, effect of ncs restraints?
Hi Ed It's very difficult to deal theoretically with NCS because, unlike bond lengths where the uncertainties are known a priori (at least in principle), with NCS you don't know the uncertainties a priori, if you see what I mean (rather like unknown unknowns!). In other words the optimal weights and hence the effective number of parameters will depend on the exactness of the NCS. In practice you can of course determine the weights by minimising Rfree w.r.t.them. So I think it would be quite difficult to do what you are proposing, i.e. to disentangle the effects of the obs/param ratio and any effect of correlation of the working test sets. Interesting problem though! BTW I think you are mis-quoting the formula in the paper, it should be Rfree/R = sqrt((Nobs+Nparam)/(Nobs-Nparam)). In other words R is reduced below its expected value in the absence of random error, by overfitting the errors in the working set, but people tend to forget that the test set also has, on average, random errors of the same magnitude which tend to increase Rfree *above* its expected value. Cheers -- Ian On Fri, Apr 9, 2010 at 8:25 PM, Edward A. Berry ber...@upstate.edu wrote: Has anyone looked theoretically at how ncs-restraints affect the expected Rfree/R ratio? Tickle et al., Acta Cryst. (1998). D54, 547-557 concluded Rfree/R = sqrt(Nobs/(Nobs-Nparam)) . He suggested that, with restrained refinement of coordinates plus individual isotropic B-factors, the effective number of parameters per atom is two. If we add strong N-fold NCS restraints on coordinates and B-factor, does that effectively reduce the number of parameters by a factor of N? Giving 2/N for parameters per atom? I'm curious how much of the drop in the r-free ratio observed on enforcing NCS is due to the reduction in the effective number of parameters, and how much is due to linking reflections in the free set with the working set. Given an expression to predict the effect of reducing number of parameters, seeing how much of the actual drop in Rfree/R it accounts for would let us see how severe the linkage problem is. Ed
Re: [ccp4bb] fink update
I think your xcode is out dated. You can download the latest one for free by login into http://connect.apple.com. Its under the Developer Tools heading. SSSRaj http://www.careforlearning.org Let us make learning a fun to everyone --- On Fri, 4/9/10, Raja Dey deyra...@yahoo.co.in wrote: From: Raja Dey deyra...@yahoo.co.in Subject: [ccp4bb] fink update To: CCP4BB@JISCMAIL.AC.UK Date: Friday, April 9, 2010, 3:21 PM Hi, Sorry for a non-ccp4 question. I hope there is someone who can solve the problem. I was trying to update fink and I got the following error. rajadey$ fink update-all Password: Information about packages read in 2 seconds. fink needs help picking an alternative to satisfy a virtual dependency. The candidates: (1) db48-aes: Berkeley DB embedded database - crypto (2) db48: Berkeley DB embedded database - non crypto Pick one: [1] Can't resolve dependency xcode (= 3.1.2) for package gcc44-4.4.2-1000 (no matching packages/versions found) Exiting with failure. Should I select the non crypto candidate? Thanking you in advance... Raja
Re: [ccp4bb] fink update
You can download the latest download from developer.apple.com/technologies/xcode.htm. The latest version is Xcode 3.2.2 and iPhone SDK 3.2Apr 3, 2010 SSSRaj http://www.careforlearning.org Let us make learning a fun to everyone --- On Fri, 4/9/10, Raja Dey deyra...@yahoo.co.in wrote: From: Raja Dey deyra...@yahoo.co.in Subject: [ccp4bb] fink update To: CCP4BB@JISCMAIL.AC.UK Date: Friday, April 9, 2010, 3:21 PM Hi, Sorry for a non-ccp4 question. I hope there is someone who can solve the problem. I was trying to update fink and I got the following error. rajadey$ fink update-all Password: Information about packages read in 2 seconds. fink needs help picking an alternative to satisfy a virtual dependency. The candidates: (1) db48-aes: Berkeley DB embedded database - crypto (2) db48: Berkeley DB embedded database - non crypto Pick one: [1] Can't resolve dependency xcode (= 3.1.2) for package gcc44-4.4.2-1000 (no matching packages/versions found) Exiting with failure. Should I select the non crypto candidate? Thanking you in advance... Raja
Re: [ccp4bb] R-free ratio, effect of ncs restraints?
Hi All, in a paper (which I can't locate now...) which I read recently it was stated that restraints do not reduce the number of parameters, rather they augment the number of data points (so strong restraints are like strong data, weak restraints weak data...). Only strict NCS constraints, where the copies have to stay exactly the same, would reduce the number of parameters. Both augment the data to parameter ratio, of course. I really liked this explanation. Mark On 9 April 2010 21:54, Ian Tickle ianj...@gmail.com wrote: Hi Ed It's very difficult to deal theoretically with NCS because, unlike bond lengths where the uncertainties are known a priori (at least in principle), with NCS you don't know the uncertainties a priori, if you see what I mean (rather like unknown unknowns!). In other words the optimal weights and hence the effective number of parameters will depend on the exactness of the NCS. In practice you can of course determine the weights by minimising Rfree w.r.t.them. So I think it would be quite difficult to do what you are proposing, i.e. to disentangle the effects of the obs/param ratio and any effect of correlation of the working test sets. Interesting problem though! BTW I think you are mis-quoting the formula in the paper, it should be Rfree/R = sqrt((Nobs+Nparam)/(Nobs-Nparam)). In other words R is reduced below its expected value in the absence of random error, by overfitting the errors in the working set, but people tend to forget that the test set also has, on average, random errors of the same magnitude which tend to increase Rfree *above* its expected value. Cheers -- Ian On Fri, Apr 9, 2010 at 8:25 PM, Edward A. Berry ber...@upstate.edu wrote: Has anyone looked theoretically at how ncs-restraints affect the expected Rfree/R ratio? Tickle et al., Acta Cryst. (1998). D54, 547-557 concluded Rfree/R = sqrt(Nobs/(Nobs-Nparam)) . He suggested that, with restrained refinement of coordinates plus individual isotropic B-factors, the effective number of parameters per atom is two. If we add strong N-fold NCS restraints on coordinates and B-factor, does that effectively reduce the number of parameters by a factor of N? Giving 2/N for parameters per atom? I'm curious how much of the drop in the r-free ratio observed on enforcing NCS is due to the reduction in the effective number of parameters, and how much is due to linking reflections in the free set with the working set. Given an expression to predict the effect of reducing number of parameters, seeing how much of the actual drop in Rfree/R it accounts for would let us see how severe the linkage problem is. Ed -- Mark J van Raaij http://webspersoais.usc.es/mark.vanraaij http://www.ibmb.csic.es
Re: [ccp4bb] fink update
Well, I have mac osx 10.5.8 and the latest Xcode available is X11- 2.5.0. To get the Xcode 3.2.2, do I have to go with OSX 10.6.X? I am wondering if that might cause problem to run programs already installed in 10.5.X. Raja From: S. Shunmugasundararaj raj_...@yahoo.com To: Raja Dey deyra...@yahoo.co.in; ccp...@dl.ac.uk Sent: Fri, 9 April, 2010 12:44:56 PM Subject: Re: [ccp4bb] fink update You can download the latest download from developer.apple.com/technologies/xcode.htm. The latest version is * Xcode 3.2.2 and iPhone SDK 3.2Apr 3, 2010 SSSRaj http://www.careforlearning.org Let us make learning a fun to everyone --- On Fri, 4/9/10, Raja Dey deyra...@yahoo.co.in wrote: From: Raja Dey deyra...@yahoo.co.in Subject: [ccp4bb] fink update To: CCP4BB@JISCMAIL.AC.UK Date: Friday, April 9, 2010, 3:21 PM Hi, Sorry for a non-ccp4 question. I hope there is someone who can solve the problem. I was trying to update fink and I got the following error. rajadey$ fink update-all Password: Information about packages read in 2 seconds. fink needs help picking an alternative to satisfy a virtual dependency. The candidates: (1)db48-aes: Berkeley DB embedded database - crypto (2)db48: Berkeley DB embedded database - non crypto Pick one: [1] Can't resolve dependency xcode (= 3.1.2) for package gcc44-4.4.2-1000 (no matching packages/versions found) Exiting with failure. Should I select the non crypto candidate? Thanking you in advance... Raja
Re: [ccp4bb] Possible sulphate
You may also want to look at some other structures at similar resolution and see what the density and interactions for their sulfates look like. E.g., if you go to HIC-Up, get to the SO4 page and then click on the link to EDS statistics - http://xray.bmc.uu.se/hicup/SO4/so4_eds_stats.html - it will show you statistics for real-space R etc. for sulfates in various resolution bins. If you go to the entry with the lowest RSR for your resolution bin, you should get an example of a well-fitting sulfate - if you pick the highest RSR you will probably find a pretty dodgy one. Click on the links to get to the EDS entries and inspect the density of the sulfates. --dvd On Wed, 7 Apr 2010, Vellieux Frederic wrote: Rex Palmer wrote: What seems to be a possible sulphate has been identified in our electron density. What steps could/should be taken to confirm or consolidate this assignment that would satisfy referees? Rex Palmer Birkbeck College Geometry of the interactions (and the shape of the electron density). Anomalous map. Even if you have only diffraction data collected at low wavelength you can always compute an anomalous map and see if the sulphur shows up. Fred. Best wishes, --Gerard ** Gerard J. Kleywegt Dept. of Cell Molecular Biology University of Uppsala Biomedical Centre Box 596 SE-751 24 Uppsala SWEDEN http://xray.bmc.uu.se/gerard/ mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. **
Re: [ccp4bb] fink update
Sorry for the confusion. It has been solved. Thanks for your help. Raja From: S. Shunmugasundararaj raj_...@yahoo.com To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 9 April, 2010 1:11:27 PM Subject: Re: [ccp4bb] fink update I think your xcode is out dated. You can download the latest one for free by login into http://connect.apple.com. Its under the Developer Tools heading. SSSRaj http://www.careforlearning.org Let us make learning a fun to everyone --- On Fri, 4/9/10, Raja Dey deyra...@yahoo.co.in wrote: From: Raja Dey deyra...@yahoo.co.in Subject: [ccp4bb] fink update To: CCP4BB@JISCMAIL.AC.UK Date: Friday, April 9, 2010, 3:21 PM Hi, Sorry for a non-ccp4 question. I hope there is someone who can solve the problem. I was trying to update fink and I got the following error. rajadey$ fink update-all Password: Information about packages read in 2 seconds. fink needs help picking an alternative to satisfy a virtual dependency. The candidates: (1)db48-aes: Berkeley DB embedded database - crypto (2)db48: Berkeley DB embedded database - non crypto Pick one: [1] Can't resolve dependency xcode (= 3.1.2) for package gcc44-4.4.2-1000 (no matching packages/versions found) Exiting with failure. Should I select the non crypto candidate? Thanking you in advance... Raja
Re: [ccp4bb] R-free ratio, effect of ncs restraints?
Hi Mark I think you need to distinguish between the mechanics of the refinement software on the one hand and the effect on the statistics on the other. I think you are referring to the former, in other words in the software the restraints are as you say treated exactly like the X-ray observations; they appear to augment the observations, and clearly do not reduce the *actual* number of parameters in any way (unlike constraints which do). Unfortunately this train of thought leads nowhere because even though in the software restraints and observations appear to be equivalent, 1 restraint is in no way *statistically* equivalent to 1 X-ray observation. We can make progress in understanding the statistics however if we consider the *effective* number of parameters which turns out to be (see the paper that Ed referred to for the proof): m_eff = m - r + Drest where m is the actual number of parameters, r is the number of restraints and Drest is a kind of correction for the fact that 1 parameter is not equivalent to 1 restraint (Drest depends on r in a complicated way; it's actually the contribution of the restraints to the least-squares residual, or equivalently to the negative log-likelihood, so 'good' restraints increase Drest less than 'bad' ones). In other words adding restraints does indeed have the effect of reducing the *effective* number of parameters (though not 1-for-1 since Drest also varies as you add restraints). We need m_eff in order to compute the ratio (no of observations) / (effective no of parameters). The expected Rfree/R (i.e. the expectation is predicated on the assumption that the parameter refinement is at a global minimum whose position in parameter space is a function of the weights you used) is then sqrt((f + m_eff) / (f - m_eff)), where f is the size of the working set. This can be written as sqrt((x+1) / (x-1)) where x = f / m_eff i.e. the effective obs/param ratio. This shows the direct relationship between Rfree/R and the effective obs/param ratio; for example you can see what happens as x tends towards unity on the one hand and towards infinity on the other! Cheers -- Ian On Fri, Apr 9, 2010 at 9:31 PM, Mark J. van Raaij mvr...@ibmb.csic.es wrote: Hi All, in a paper (which I can't locate now...) which I read recently it was stated that restraints do not reduce the number of parameters, rather they augment the number of data points (so strong restraints are like strong data, weak restraints weak data...). Only strict NCS constraints, where the copies have to stay exactly the same, would reduce the number of parameters. Both augment the data to parameter ratio, of course. I really liked this explanation. Mark On 9 April 2010 21:54, Ian Tickle ianj...@gmail.com wrote: Hi Ed It's very difficult to deal theoretically with NCS because, unlike bond lengths where the uncertainties are known a priori (at least in principle), with NCS you don't know the uncertainties a priori, if you see what I mean (rather like unknown unknowns!). In other words the optimal weights and hence the effective number of parameters will depend on the exactness of the NCS. In practice you can of course determine the weights by minimising Rfree w.r.t.them. So I think it would be quite difficult to do what you are proposing, i.e. to disentangle the effects of the obs/param ratio and any effect of correlation of the working test sets. Interesting problem though! BTW I think you are mis-quoting the formula in the paper, it should be Rfree/R = sqrt((Nobs+Nparam)/(Nobs-Nparam)). In other words R is reduced below its expected value in the absence of random error, by overfitting the errors in the working set, but people tend to forget that the test set also has, on average, random errors of the same magnitude which tend to increase Rfree *above* its expected value. Cheers -- Ian On Fri, Apr 9, 2010 at 8:25 PM, Edward A. Berry ber...@upstate.edu wrote: Has anyone looked theoretically at how ncs-restraints affect the expected Rfree/R ratio? Tickle et al., Acta Cryst. (1998). D54, 547-557 concluded Rfree/R = sqrt(Nobs/(Nobs-Nparam)) . He suggested that, with restrained refinement of coordinates plus individual isotropic B-factors, the effective number of parameters per atom is two. If we add strong N-fold NCS restraints on coordinates and B-factor, does that effectively reduce the number of parameters by a factor of N? Giving 2/N for parameters per atom? I'm curious how much of the drop in the r-free ratio observed on enforcing NCS is due to the reduction in the effective number of parameters, and how much is due to linking reflections in the free set with the working set. Given an expression to predict the effect of reducing number of parameters, seeing how much of the actual drop in Rfree/R it accounts for would let us see how severe the linkage problem is. Ed -- Mark J van Raaij
