Re: [ccp4bb] Protein-detergent micelle sizes
In case you end up compiling your own list, here is one entry: von Jagow and co-workers (Biochim Biophys Acta. 1977 462(3):549-58.) used tritiated Triton X-100 to measure the binding to Complex III 5. In accordance with the high polarity the amount of bound detergent is relatively low, it amounts to 0.2 g Triton X-100/g protein. The amount of Triton bound to 1 mol of b-c1-dimer corresponds to the molecular weight of the Triton micelle. The bc1 dimer MW is ~480 kDa, so .2 g/g - 96 kDa detergent Jacob Keller wrote: Dear Crystallographers, I am trying to gather literature values on the size of the detergent/micelle belts on solublized membrane proteins. Does anybody know of a good repository of this info, whether as a database or as a review paper, or otherwise? The rough values of 15-25kD to 35-50kD were given in one recent BB posting, but I was looking for a more systematic tabulation... Thanks, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
[ccp4bb] Very low B factor
Dear All, I am refining a low resolution structure of 3 Angstrom. I refined the structure to 25 R value and 29 R free. But some of the residues are showing very low B factors (less than 5).Please advice me how to tackle this problem. As mentioned in a previous post I used babinet scaling in Refamc but still it is the same. Thank you in advance. Jobi
Re: [ccp4bb] Protein-detergent micelle sizes
Jacob, Savvas' suggestion about the the Calbiochem and Anatrace product catalogs is a good one, particularly as the latter is fairly up to date regarding the newest detergents. Reviews tend to be out of date quickly. Also, concerning methods and the odd useful measurements of detergent- binding to membrane proteins, get almost any paper written by Marc le Maire on the subject. Finally, since everyone now has access to analytical ultracentrifugation, consider doing AUC with density matching with D2O, sucrose, or Nycodenz. Ariel Lustig's paper on the subject (Biochimica et Biophysica Acta 1464, 199-206, 2000) is an excellent guide to the method, although it took nearly 20 years to get him to publish his Model E data. A lot of his useful measurements were put in diplom and Ph.D. theses at the Biozentrum and virtually forgotten. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Nov 20, 2007, at 4:22 AM, Edward Berry wrote: In case you end up compiling your own list, here is one entry: von Jagow and co-workers (Biochim Biophys Acta. 1977 462(3):549-58.) used tritiated Triton X-100 to measure the binding to Complex III 5. In accordance with the high polarity the amount of bound detergent is relatively low, it amounts to 0.2 g Triton X-100/g protein. The amount of Triton bound to 1 mol of b-c1-dimer corresponds to the molecular weight of the Triton micelle. The bc1 dimer MW is ~480 kDa, so .2 g/g - 96 kDa detergent Jacob Keller wrote: Dear Crystallographers, I am trying to gather literature values on the size of the detergent/micelle belts on solublized membrane proteins. Does anybody know of a good repository of this info, whether as a database or as a review paper, or otherwise? The rough values of 15-25kD to 35-50kD were given in one recent BB posting, but I was looking for a more systematic tabulation... Thanks, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
Re: [ccp4bb] Protein-detergent micelle sizes
Jacob, Whether the calbiochem/anatrace catalogues will work for you depends on what you want to know. They do have useful info on micelle sizes (aggregation #s, cmc's etc), but these are values for micelles alone, either in water or 0.1 M salt or something like that. If your question is how much detergent is actually associated with a particular protein you'll unfortunately have to measure that yourself (see Michaels message), since this will be protein and condition dependent (ie a large membrane protein will have more detergent associated with it than a small one). There was a thread on this subject on this board a couple weeks back i think. Cheers, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of Savvas Savvides Sent: Tue 11/20/2007 3:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein-detergent micelle sizes Hi Jacob, the Calbiochem and Anatrace product catalogues will do the trick for you. best wishes Savvas Savvas N. Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19 Email: [EMAIL PROTECTED] http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Tuesday, November 20, 2007 12:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein-detergent micelle sizes Dear Crystallographers, I am trying to gather literature values on the size of the detergent/micelle belts on solublized membrane proteins. Does anybody know of a good repository of this info, whether as a database or as a review paper, or otherwise? The rough values of 15-25kD to 35-50kD were given in one recent BB posting, but I was looking for a more systematic tabulation... Thanks, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
[ccp4bb] [SUMMARY]: Twinning problem
To the CCP4 community, I received a number of very helpful replies to my lengthy question last week about twinning and/or problems with indexing. I am very grateful for all the suggestions and I have learned more about what to look for and what to try. You will find the replies (chronological order) below and my original question at the bottom. Thank you, -Andy Torelli Daniel Schlieper wrote: In addition to the twinning tests, it might be useful to look at the reflections in pseudo-precession images (eg, hklview). Check if the systematically absent reflections behave like predicted. Eleanor Dodson wrote: You can read the documentation on twinning http://www.ccp4.ac.uk/dist/html/twinning.html You can have twinning in I23 and the best indicator is that table of I^2 /I^2 That looks significantly 2 so it is likely you do have twinned data. The twinning would make hkl nearly equal to k h -l, (a symmetry pair for I423) so you would not get very good stats in I423 Seeing it is likely to be I23 - not I423 - you also have to be careful when merging crystals since there are two equally valid ways to index any crystal (see http://www.ccp4.ac.uk/dist/html/reindexing.html fo some discussion) There is a program pointless written by Phil Evans in the latest CCP4 release. That allows you to give a master data set - I woud choose the least twinned - and suggested reindexing all new crystals to the first chosen convention. It will also tell you how strong the 4-fold symmetry that is generated by the twinning) and give you a preliminary indication of the amount of twinning. Perfect 4-fold = perfect twinning ( 0.5 twin factor) You can use it with mosflm output, scalepack unmerged output or xds ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/pointless-1.2.9.tar.gz and pointless.html for documentation. Peter Zwart suggested downloading Phenix and running phenix.xtriage. Graeme Winter wrote: I would start at the beginning - how sure are you of the lattice? You say that the indexing solutions are poor. A very useful thing I find is to compare the statistics from postrefinement with the asserted lattice applied and ignored - roughly speaking refine the cell in I23 / I4 and separately in P1 and see if the stats (r.m.s. deviations etc) look much better in P1. If they do, I would take a closer look, perhaps testing lower symmetry lattices. This is something that xia2 does, so perhaps you could give this a go (had to give it a plug somewhere!) If the stats come out about the same (this test is more sensitive to cell angles than cell lengths, so is unlikely to distinguish between your two candidate spacegroups. What I would then to is take the integrated but unscaled reflection file and put this into something like pointless or xtriage and see what symmetry elements they find - this should give you a pretty solid (twinning notwithstanding) determination of the correct pointgroup. From here the twinning tests should be more helpful. Miroslav Papiz wrote: Irrespective of twinning issues. Have you looked at I432 ? if you are convinced that the 4 and 3 folds are present (i.e similar Rsym in I422 and I23) then this is also a possible spacegroup. You should at least try this before looking for other explanations. Bill Scott wrote: I can say that phenix is very good at handling both twinning and RNA. phenix.xtriage will also give you useful information about twinning (applies several tests, etc) and the phenix authors have been incredibly helpful and are in fact looking for RNA test cases, so it would be easy to seek professional advice. Dominika Borek wrote: Answers to your questions depend a lot on other things: Twinning alone: 1. Are you going use molecular replacement or rather heavy atoms-based phasing? 2. How many copies of RNA molecule do you expect in your ASU (quite large BTW)? 3. If you plan to phase with HA - which HA are you using or going to use? Data alone: 1. Did you collect at home or at synchrotron? I expect that with such large unit cell you collected at synchrotron. If this is the case and if you have in your crystallization solution (as it happens for RNA) potassium, manganese, rubidium etc. radiation damage even at home source will be the issue. I do not know what software did you used. If it was scalepack and you used option fit B during scaling, log file will tell you quite well how bad was radiation damage. If fitted B-factor is higher than 5-6 for the last images that already indicates significant radiation damage. Another indication of radiation damage is exactly what you described - high R-merge, a lot of rejections etc. Unfortunately, to tell more I would have to see .log file from scaling/merging software. I personally think that it is quite possible that you do not have twinning just lower symmetry (completeness issue suggests it). Lack of birefringence
Re: [ccp4bb] Unmerged output from Scala
It is worth adding a note that if you are remerging data it is also a great idea to have sdcorrection noadjust 1.0 0.0 somewhere in your script so that the errors are not inflated a second time by the default values (sdadd of 0.02) - I am pretty sure that this is necessary, certainly seemed to be. Yes, people appear to do this. I hadn't been bitten by this one quite yet as I have been using ONLYMERGE RESTORE with the SCALES file and rescaling / remerging the data but without recalculating the scales. Seems like this wasn't as bad an idea as I thought it was ;o) Perhaps naming the SCALE columns differently when they have been applied so that they are not reapplied would be a good idea, something like SCALEUSED. This takes us back to the beginning of this thread, always fun. Having output of both merged and unmerged reflection files by default could be a cause for some confusion if someone tries to take one of the unmerged files and put it into truncate... Bang! Cheers, Graeme -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Phil Evans Sent: 20 November 2007 15:51 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Unmerged output from Scala If you don't put INSCALE OFF, the scales will be applied a second time you will get (much) worse data I didn't know anyone was doing this You should get the same statistics (Rmerge etc) if you reinput an OUTPUT UNMERGED file with ONLYMERGE; INSCALE OFF I'll look into how complicated it would be to write both files (but if you have eg multi-wavelength you might get a lot of files out :-( ) Phil On 20 Nov 2007, at 15:44, Frank von Delft wrote: Does it make a big difference if you *don't* have INSCALE OFF? I just notice I've been doing it for years. I use OUTPUT UNMERGED as well in my standard scaling script, in which I run two scalas in sequence: the first writes out the unmerged data and scales, the second only merges. The purpose is to have both unmerged and merged data after I've scaled, the former for xprep, pointless etc, the latter for sharp, refinement, etc. If scala could dump out both versions I wouldn't bother either. phx. Clemens Vonrhein wrote: Hi Graeme, even with these options (ONLYMERGE and SCALES CONSTANT) you will have the SCALE column applied again to the intensities (not good) - at least that's how I understood Phil. You need to use ONLYMERGE INSCALE OFF to use the already scaled intensities and avoid applying the SCALE column again - Phil was the one telling me that. I was scratching my head for a long time trying to understand those various INTIAL/ONLYMERGE/INSCALE/NOSCALE options and how they relate to each other ... Cheers Clemens On Mon, Nov 19, 2007 at 08:07:02PM -, Winter, G (Graeme) wrote: Hi Phil, I use this option but not these columns. The only time I feed the file back I use ONLYMERGE and SCALES CONSTANT, to remerge the reflections. Cheers, Graeme From: CCP4 bulletin board on behalf of Phil Evans Sent: Mon 19/11/2007 5:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Unmerged output from Scala Is anyone using the OUTPUT UNMERGED option in Scala? This file contains columns called SCALE SIGSCALE which are the applied scale and its SD I propose to change the names of these columns so that if you put the file back into Scala the scales do not get re-applied by default (which is wrong since they have been applied already) Will this cause anyone problems? I suspect that very few people or programs are using this file Phil Evans
Re: [ccp4bb] Unmerged output from Scala
If you don't put INSCALE OFF, the scales will be applied a second time you will get (much) worse data I didn't know anyone was doing this You should get the same statistics (Rmerge etc) if you reinput an OUTPUT UNMERGED file with ONLYMERGE; INSCALE OFF I'll look into how complicated it would be to write both files (but if you have eg multi-wavelength you might get a lot of files out :-( ) Phil On 20 Nov 2007, at 15:44, Frank von Delft wrote: Does it make a big difference if you *don't* have INSCALE OFF? I just notice I've been doing it for years. I use OUTPUT UNMERGED as well in my standard scaling script, in which I run two scalas in sequence: the first writes out the unmerged data and scales, the second only merges. The purpose is to have both unmerged and merged data after I've scaled, the former for xprep, pointless etc, the latter for sharp, refinement, etc. If scala could dump out both versions I wouldn't bother either. phx. Clemens Vonrhein wrote: Hi Graeme, even with these options (ONLYMERGE and SCALES CONSTANT) you will have the SCALE column applied again to the intensities (not good) - at least that's how I understood Phil. You need to use ONLYMERGE INSCALE OFF to use the already scaled intensities and avoid applying the SCALE column again - Phil was the one telling me that. I was scratching my head for a long time trying to understand those various INTIAL/ONLYMERGE/INSCALE/NOSCALE options and how they relate to each other ... Cheers Clemens On Mon, Nov 19, 2007 at 08:07:02PM -, Winter, G (Graeme) wrote: Hi Phil, I use this option but not these columns. The only time I feed the file back I use ONLYMERGE and SCALES CONSTANT, to remerge the reflections. Cheers, Graeme From: CCP4 bulletin board on behalf of Phil Evans Sent: Mon 19/11/2007 5:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Unmerged output from Scala Is anyone using the OUTPUT UNMERGED option in Scala? This file contains columns called SCALE SIGSCALE which are the applied scale and its SD I propose to change the names of these columns so that if you put the file back into Scala the scales do not get re-applied by default (which is wrong since they have been applied already) Will this cause anyone problems? I suspect that very few people or programs are using this file Phil Evans
Re: [ccp4bb] Unmerged output from Scala
Does it make a big difference if you *don't* have INSCALE OFF? I just notice I've been doing it for years. I use OUTPUT UNMERGED as well in my standard scaling script, in which I run two scalas in sequence: the first writes out the unmerged data and scales, the second only merges. The purpose is to have both unmerged and merged data after I've scaled, the former for xprep, pointless etc, the latter for sharp, refinement, etc. If scala could dump out both versions I wouldn't bother either. phx. Clemens Vonrhein wrote: Hi Graeme, even with these options (ONLYMERGE and SCALES CONSTANT) you will have the SCALE column applied again to the intensities (not good) - at least that's how I understood Phil. You need to use ONLYMERGE INSCALE OFF to use the already scaled intensities and avoid applying the SCALE column again - Phil was the one telling me that. I was scratching my head for a long time trying to understand those various INTIAL/ONLYMERGE/INSCALE/NOSCALE options and how they relate to each other ... Cheers Clemens On Mon, Nov 19, 2007 at 08:07:02PM -, Winter, G (Graeme) wrote: Hi Phil, I use this option but not these columns. The only time I feed the file back I use ONLYMERGE and SCALES CONSTANT, to remerge the reflections. Cheers, Graeme From: CCP4 bulletin board on behalf of Phil Evans Sent: Mon 19/11/2007 5:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Unmerged output from Scala Is anyone using the OUTPUT UNMERGED option in Scala? This file contains columns called SCALE SIGSCALE which are the applied scale and its SD I propose to change the names of these columns so that if you put the file back into Scala the scales do not get re-applied by default (which is wrong since they have been applied already) Will this cause anyone problems? I suspect that very few people or programs are using this file Phil Evans
Re: [ccp4bb] Unmerged output from Scala
If you don't put INSCALE OFF, the scales will be applied a second time you will get (much) worse data I didn't know anyone was doing this You should get the same statistics (Rmerge etc) if you reinput an OUTPUT UNMERGED file with ONLYMERGE; INSCALE OFF Oh wait, I use RESTORE SCALES with ONLYMERGE, which is, I think, different to what you ask. Certainly the statistics are the same -- except intensity-based tables, mind you. Embarrassing, I never even checked. I'll look into how complicated it would be to write both files (but if you have eg multi-wavelength you might get a lot of files out :-( ) Good! Saves me having to run a lot of scala jobs. Just about every phasing program suite uses unmerged data somewhere along the line (correct me if I'm wrong), so you may as well generate it. phx.
[ccp4bb] senior scientist at the UWM
Dear Colleagues, I want to bring the following senior scientist position to your attention. Please do not reply to me but to Abbas Ourmazd directly. == THE UNIVERSITY OF WISCONSIN #8211 MILWAUKEE Senior Research Scientist The Department of Physics at the University of Wisconsin #8211 Milwaukee seeks outstanding applicants for a senior scientist position starting March 2008. Candidates must have: a PhD in physics or equivalent; at least three years of postdoctoral research experience in electron and/or X-ray scattering methods; and at least three years of independent research experience as evidenced by publications. Responsibilities include research in structure determination by scattering methods, development of advanced noise-robust algorithms for structure recovery and reconstruction, and leading research associates. Expert knowledge of relevant areas of physics and software development and management of group of software developers are essential. At least five years of complex software development and commitment to collegial research are preferred. Application deadline is Dec. 15, 2007. Applicants should submit their vita, summary of relevant research experience, publications and patents lists, and three letters of recommendation. Electronic submission is highly encouraged; please forward to Professor Abbas Ourmazd ([EMAIL PROTECTED]), with an electronic copy to Kate Valerius ([EMAIL PROTECTED]). As an equal opportunity/affirmative action employer, the University of Wisconsin #8211 Milwaukee promotes excellence through diversity and encourages all qualified individuals to apply. The names of those applicants who have not requested that their identities be withheld and the names of all finalists will be released upon request. Contact: Professor Abbas Ourmazd, Department of Physics, University of Wisconsin-Milwaukee, P.O. Box 413, Milwaukee, WI 53201 ([EMAIL PROTECTED]). For more information, please visit: www.uwm.edu/letsci/jobs/index.html. = Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: [EMAIL PROTECTED] http://users.physik.tu-muenchen.de/marius/
[ccp4bb] Installation problem on Fedora 8
I am attempting to install and compile CCP4-6.0.2 along with BLT,
Chooch, etc under Fedora 8. I am doing this under the root account, with
a default login shell of tcsh. When I run install.sh, BLT and Chooch
apear to install OK and then I get these error messages:
##
# CCP4 Installation #
##
Creating ccp4.setup file...
sed: can't read /root/ccp4-6.0.2/include/ccp4.setup-sh: No such file or
directory
./install.sh: line 477: /root/ccp4-6.0.2/include/ccp4.setup-sh: No such
file or directory
sed: can't read /root/ccp4-6.0.2/include/ccp4.setup-zsh: No such file or
directory
./install.sh: line 479: /root/ccp4-6.0.2/include/ccp4.setup-zsh: No such
file or directory
sed: can't read /root/ccp4-6.0.2/include/ccp4.setup-dist: No such file
or directory
./install.sh: line 481: /root/ccp4-6.0.2/include/ccp4.setup-dist: No
such file or directory
sed: can't read /root/ccp4-6.0.2/include/ccp4.setup-bash: No such file
or directory
./install.sh: line 483: /root/ccp4-6.0.2/include/ccp4.setup-bash: No
such file or directory
./install.sh: line 504: cd: ccp4-6.0.2: No such file or directory
cp: cannot stat `include/ccp4.setup-dist': No such file or directory
./install.sh: line 506: include/ccp4.setup-sh: No such file or directory
[EMAIL PROTECTED] ccp4_master]# pwd
/usr/local/ccp4_master
[EMAIL PROTECTED] ccp4_master]# echo $CCP4_MASTER
/usr/local/ccp4_master
--
The installation is by default supposed to happen in the current
directory, but references to the current directory in install.sh as
${current} seem to be failing, as though each command were instead being
executed from root's home directory. Help, anyone?
Cheers,
--
===
With the single exception of Cornell, there is not a college in the
United States where truth has ever been a welcome guest - R.G. Ingersoll
===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
[EMAIL PROTECTED]
