[ccp4bb] hkl2000 install
Dear everyone: I'm sorry for a little off-topic! I want to install HKL2000 on ubuntu11.10 32bits, but it produces a file named info not cr_info after run the access_prod program.And when I put info to /usr/local/lib directory and typingHKL2000 in terminal, it display: root@ubuntu:/usr/local/bin# HKL2000 ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found Error code: -1 So could anyone tell me how to do it ? Rui Wang
Re: [ccp4bb] hkl2000 install
王瑞 wangrui...@gmail.com writes: I'm sorry for a little off-topic! I want to install HKL2000 on ubuntu11.10 32bits, but it produces a file named info not cr_info after run the access_prod program.And when I put info to /usr/local/lib directory and typingHKL2000 in terminal, it display: root@ubuntu:/usr/local/bin# HKL2000 ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found Error code: -1 So could anyone tell me how to do it ? Yes, you use your “info” file to obtain a “cr_info” file from HKL Research: http://www.hkl-xray.com/ Regards, -- Ian ◎
[ccp4bb] usefulness of cacodylate?
Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx
Re: [ccp4bb] hkl2000 install
info is send to HKL.com to get cr_info if you have a legal version FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 9, 2012, at 12:37 , 王瑞 wangrui...@gmail.com wrote: Dear everyone: I'm sorry for a little off-topic! I want to install HKL2000 on ubuntu11.10 32bits, but it produces a file named info not cr_info after run the access_prod program.And when I put info to /usr/local/lib directory and typingHKL2000 in terminal, it display: root@ubuntu:/usr/local/bin# HKL2000 ERROR: Not a valid HKL-2000 license: Licence info file (cr_info) not found Error code: -1 So could anyone tell me how to do it ? Rui Wang
Re: [ccp4bb] usefulness of cacodylate?
Hi, I guess Cacodylate is safe as long as you don't come into direct contact with it, or dispose it off in a careless fashion. Aren't there substances in biochemical labs which are a equally if not more harmful, like acryamide or Ethidium Bromide, for instance? But I agree that it should be done away with if possible, and in the case of Cacodylate there is an equally effective and much safer option in MES. regards Ganesh Le 09/11/12 13:26, Frank von Delft a écrit : Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx
Re: [ccp4bb] usefulness of cacodylate?
Hi Frank, I worked with protein purification buffers and crystallization buffers containing 20mM potassium cacodylate for five years or so. And yes, the only precaution I used was gloves while weighing the chemical and while making buffers etc. And not just me, but all of my former colleagues worked with cacodylate. Then, there's those nasty chemicals for heavy atom soaks like mercury and tantalum compounds that are equally hazardous. And... many other chemicals we used on a daily basis in the lab but we don't suspect as much as we should. Nucleosome core particles stubbornly refuse to crystallize if you leave out cacodylate, not just from the crystallization buffers but also from the protein purification buffers. Yes, there are folks who accidentally left it out and never gotten crystals of nucleosomes. I don't rule out that someday someone may very well be able to substitute cacodylate for some other chemical and successfully crystallize nucleosomes. It might be hard to interpret the kind of studies you suggest and here's why, in my opinion. Even if one showed that there was no need for cacodylate for, say, a 1000 different proteins, I would definitely not exclude it from a crystallization screen for my favorite protein because we have not gotten to that point in crystallography where one can predict crystallization conditions for a new macromolecule with great accuracy. In my opinion, it's all relative. There are probably more chances of me being killed by a reckless bicyclist in Boston/Cambridge than by cacodylate. ;-) Cheerios! Raji On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.**com/doi/10./j.1365-2818.** 1977.tb01136.x/abstracthttp://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.**gov/chemical/4468http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] usefulness of cacodylate?
Hi Frank, As the old saying goes: it's all relative (maybe not so old actually, it's only since Einstein's relativity theory, right?). Cacodylate was used, among other things, in buffers as a substitute for phosphate, merely because it kills bugs. Nowadays, we use azide, which is just as hazardous (well maybe not, if you read the details on the bottle) but still not recommended. And as Raji and others have pointed out, our heavy atom compounds, acrylamide, ethidium bromide, you name them, are not nice either. We have to take precautions of all sorts and I guess that's what your review committee will be looking for. I'd give them the credit that they're sensible people, unlike our University administration that declare ALL chemicals as dangerous (regardless of the data sheet), including NaCl, glucose, Superdex columns etc. whatever we order, just so that they can charge us 1.5% extra on the price (which is already much higher than what you pay) as we order the goods, all under the pretext that it's for disposing of the material out of the lab some time in the future (of course Superdex columns are never being disposed, and so is NaCl which is used to the last grain). Meanwhile, the admins can use the money for their needs. Clever people, right? Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Friday, November 09, 2012 3:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] usefulness of cacodylate? Hi Frank, I worked with protein purification buffers and crystallization buffers containing 20mM potassium cacodylate for five years or so. And yes, the only precaution I used was gloves while weighing the chemical and while making buffers etc. And not just me, but all of my former colleagues worked with cacodylate. Then, there's those nasty chemicals for heavy atom soaks likemercury and tantalum compounds that are equally hazardous. And... many other chemicals we used on a daily basis in the lab but we don't suspect as much as we should. Nucleosome core particles stubbornly refuse to crystallize if you leave out cacodylate, not just from the crystallization buffers but also from the protein purification buffers. Yes, there are folks who accidentally left it out and never gotten crystals of nucleosomes. I don't rule out that someday someone may very well be able to substitute cacodylate for some other chemical and successfully crystallize nucleosomes. It might be hard to interpret the kind of studies you suggest and here's why, in my opinion. Even if one showed that there was no need for cacodylate for, say, a 1000 different proteins, I would definitely not exclude it from a crystallization screen for my favorite protein because we have not gotten to that point in crystallography where one can predict crystallization conditions for a new macromolecule with great accuracy. In my opinion, it's all relative. There are probably more chances of me being killed by a reckless bicyclist in Boston/Cambridge than by cacodylate. ;-) Cheerios! Raji On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] model protein for ligand soaking
Hi All, For a tutorial, we would like to demonstrate the method of ligand soaking in protein crystals. I am thinking about using some sort of native ligand or inhibitor that can be easily identified in the electron density rather than halide or heavy atom derivatization. Does anybody have a suggestion for a protein/ligand combination that could be used for that and that is commercially available? Thanks! Uli -- Ulrich Zander High Troughput Crystallization Lab/Diffraction Instrumentation Team EMBL Grenoble Outstation 6 rue Jules Horowitz 38000 Grenoble, France Phone: +33 47620-9487
Re: [ccp4bb] usefulness of cacodylate?
Well originally we got these in cacodylate and not much else would diffract. http://www.pdb.org/pdb/explore/materialsAndMethods.do?structureId=2EPH But nowadays I have another conditions, which does not require cacodylate and works well with Hepes. Jürgen On Nov 9, 2012, at 7:26 AM, Frank von Delft wrote: Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] usefulness of cacodylate?
Hi Frank, In our hands, some RNAs only crystallize out of cacodylate buffers. We would otherwise stop using it out of health and safety concerns. Blaine Blaine Mooers Assistant Professor Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center S.L. Young Biomedical Research Center Rm. 466 Letter address: Shipping address: P.O. Box 26901, BRC 466 975 NE 10th Street, BRC 466 Oklahoma City, OK 73190 Oklahoma City, OK 73104-5419 office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910 e-mail: blaine-moo...@ouhsc.edu webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d- From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft [frank.vonde...@sgc.ox.ac.uk] Sent: Friday, November 09, 2012 6:26 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] usefulness of cacodylate? Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx
Re: [ccp4bb] model protein for ligand soaking
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Uli, the geometric ligands (I3C, B3C, B4C) by Tobias Beck (http://www.protein.ethz.ch/people/tobias/) are both commercially available and easily identified. I3C is suitable for inhouse data collection and provide a strong anomalous signal. Of course they are not 'native' but seem to suit your needs. Cheers, Tim On 11/09/2012 03:12 PM, Ulrich Zander wrote: Hi All, For a tutorial, we would like to demonstrate the method of ligand soaking in protein crystals. I am thinking about using some sort of native ligand or inhibitor that can be easily identified in the electron density rather than halide or heavy atom derivatization. Does anybody have a suggestion for a protein/ligand combination that could be used for that and that is commercially available? Thanks! Uli - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQnRbMUxlJ7aRr7hoRApQnAKCucaAAIxBnZ7dAkSwaUn/mh81ElgCgi1Ju 15N3YQcFJThqxTrKQd9WhuA= =BGVo -END PGP SIGNATURE-
Re: [ccp4bb] model protein for ligand soaking
Human carbonic anhydrase II and any sulfonamide. We use a variety of alkylsulfonamides (everyone can have a different ligand and they are easy to model) or you can use commercially available inhibitors like benzenesulfonamide, sulfanilamide, acetazolamide, etc. A 30 second soak is sufficient to populate the active site. Kd values are in the nanomolar to picomolar range for most inhibitors. 1 mM or superstoichiometric, whichever is smaller, is sufficient. Doug Juers at Whitman does a nice teaching lab with bovine trypsin and benzamidine. I am gong to plagiarize that one (with Doug's permission) for my teaching lab when I get tired of human CA-II. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/9/2012 9:12 AM, Ulrich Zander wrote: Hi All, For a tutorial, we would like to demonstrate the method of ligand soaking in protein crystals. I am thinking about using some sort of native ligand or inhibitor that can be easily identified in the electron density rather than halide or heavy atom derivatization. Does anybody have a suggestion for a protein/ligand combination that could be used for that and that is commercially available? Thanks! Uli
Re: [ccp4bb] model protein for ligand soaking
On Fri, 2012-11-09 at 15:12 +0100, Ulrich Zander wrote: Does anybody have a suggestion for a protein/ligand combination that could be used for that and that is commercially available? Perhaps lysozyme complexed with some sugar? -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
[ccp4bb] Opportunity for postdocs (LCLS, XFEL)
PETER PAUL EWALD FELLOWSHIPS BY THE VOLKSWAGEN FOUNDATION The recent launch of X-ray free electron lasers creates unprecedented research opportunities. Aiming at an early progress of this field, the Volkswagen Foundation awards the so called Peter Paul Ewald Fellowships for projects carried out at the LCLS in Stanford, USA, in affiliation with an institute in Germany. Early phase postdoctoral researchers are invited to apply. In 2013 up to five fellowships for three years will be awarded. Next application deadline: January 25, 2013. For more information please visit our website www.volkswagenstiftung.de/ewald-fellowshipshttp://www.volkswagenstiftung.de/ewald-fellowships or contact Dr. Ulrike Bischler VolkswagenStiftung Kastanienallee 35 30519 Hannover GERMANY Phone: +49 (0) 511 8381 350 Fax: +49 (0) 511 8381 4350 bisch...@volkswagenstiftung.demailto:bisch...@volkswagenstiftung.de www.volkswagenstiftung.dehttp://www.volkswagenstiftung.de
Re: [ccp4bb] Script for removing/gathering select rows of data
Dear all, thanks for your help--I made a very simple little script using awk, and it works excellently (anyone is welcome to it, of course). Thanks so much for taking the time. Jacob On Wed, Nov 7, 2012 at 9:49 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, does anyone have a script on hand which can: -read in a tab-delimited dataset -determine whether each row in column X is above or below some input cutoff value Y, -outputs all such rows to a new file? if someone already has this on hand, I would appreciate it--seems like it should be a very generally useful script to have around, and I need such right now... Thanks very much, Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] usefulness of cacodylate?
Cacodylate, being an arsenic compound, is moderately toxic. You do have to ingest it, however, for it to be toxic. Normal lab protection should be sufficient. It is any more concerning to me than lithium salts, mercury, acrylamide, ethidium bromide, etc. Having said that, we usually abandon it as a buffer when optimizing conditions that include it. Typically, substituting 0.1 M MES pH 6.5 for cacodylate, pH 6.5, is inconsequential for crystallization for most proteins, but of course there are always going to be exceptions. Cacodylate will react with thiols, and this can complicate crystallization from solutions that contain high concentrations of DTT or beta-ME. Cacodylate can also bind to thiol groups on proteins. (This may be a plus or a minus, depending on your point of view.) Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/9/2012 7:26 AM, Frank von Delft wrote: Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx
[ccp4bb] Assemble Protein-DNA complex
Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Assemble Protein-DNA complex
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei Huang, if you are lucky you can form the complex as crystal in the drop (I assume you want to crystallise the protein-DNA complex): set up drops at high salt concentration (as low as possible to keep the protein in solution at reasonable concentration) in the presence of DNA. Prepare the buffer the same way as the protein buffer, but with reduced salt concentration (btw, you can also try divalent ions, eg. MgCl2). This way water evaporates into the drop, diluting the salt concentration. - From your discription the solubility of the protein drops faster than its concentration, hence it should precipitate. And, as I said, if you are lucky, it does so as a crystal in complex with the DNA. Best, Tim On 11/09/2012 05:14 PM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6 nvrZMfMmeekAauJ26jy6jbE= =nbV+ -END PGP SIGNATURE-
Re: [ccp4bb] Assemble Protein-DNA complex
Dear Wei, If i understand your different experiment, you try to obtain your protein DNA complex at different salt concentration with different method to reach the final concentration. I read that you try 150 mM KCl + 150 mM NaCl as high concentration salt, it result in 300 mM cations and 300 mM Cl. To my mind, and according your experiment it's too high. But when you try low concentration you dialized the protein alone in 30 and 100 mM salt concentration. In my opinion the best way, is to dialize the protein mixed with the DNA. Because the protein will probably be stablize by DNA. If the protein meet slowly DNA at the same time the salt concentration decrease, you minimize the precipitation (it's your third experiment in fact). You have not precised the final salt concentration with this method. If you try 30 mM, maybe it's too low, i suggest 100mM Salt. I remember paper about nucleosome where author explain solution with multiples steps dializis. It's more gradual and it could help if you want reach very low salt concentration. One other idea, is to play with the pH, because of the protein's pI. In function of the pI you have to try different pH for the complex preraration. You can optimise the protein DNA interaction through the protein charge. HtH Nicolas Le 09/11/12 17:14, Wei Huang a écrit : Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] usefulness of cacodylate?
Math may be frightening but cacodylate seems not... With a MW of 214 for the trihydrate a 70 kg clone needs at the 0.5 g/kg LD50 to consume about 35 g of it, which is 0.16M. Of a 0.1M solution you'd therefore have to drink 1.6 L or almost 4 pints. So, prost, cheers, gsuffa, bescheid, slantje, na strovje etc! BR PS: it is really not 0.5mg/g for the LD50 - it is indeed 0.25 to 0.5 g/L - I checked. For waterflea it is lower though... PPS: I remember with horror from my inorganic Chemistry Lab taking place in Boltzmann's Labs (and with the same curriculum as I suspect now), a smell test (as in sniffing into the test tube) for As, called the Cacodylprobe (aka Krokodilprobe). As I am still around (although the As sniffing may explain a few things) panic appears unwarranted. - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ --- The road to scientific serfdom is paved with Nature papers --- -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Friday, November 09, 2012 4:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] usefulness of cacodylate? Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstra ct and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx
Re: [ccp4bb] Assemble Protein-DNA complex
This sounds like a job for ammonium acetate. Use it as your salt. Purify your complex in it and then set up drops where they wells have the amount of ammonium acetate needed to keep your protein stable and the wells have none, or a range of concentrations. The ammonium acetate will equilibrate by vapor diffusion, lowering the concentration in the drop and causing your complex to come out of solution. James On Nov 9, 2012, at 9:14 AM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Assemble Protein-DNA complex
I meant where the drops have the concentration of ammonium acetate needed. James On Nov 9, 2012, at 10:06 AM, James Stroud wrote: This sounds like a job for ammonium acetate. Use it as your salt. Purify your complex in it and then set up drops where they wells have the amount of ammonium acetate needed to keep your protein stable and the wells have none, or a range of concentrations. The ammonium acetate will equilibrate by vapor diffusion, lowering the concentration in the drop and causing your complex to come out of solution. James On Nov 9, 2012, at 9:14 AM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] low-resolution and zinc
What program do you use for refinement? FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 9, 2012, at 20:09 , SD Y ccp4...@hotmail.com wrote: Dear All, Thanks for all the suggestions on low resolution, SG and Zn and I learned a lot. I am not done yet. I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger Rowlett. Also an excellent protocol is available at http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol seems to be very straight forward. I am not getting the result I wanted. 1. When ever I generate cif file, its not writing any for ZN-SG links at all. 2. After refmac refinement it only draw in LINK for His-ZN. Please see the images https://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.png https://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png 3. I get this His-Zn link without using .cif file, so what stage do I use this .cif file? 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing LINK in PDB. I only see LINKR ZNZN H 1 SG CYS A 83ZN-CYS I dont know what is missing, I also attached the log file which generated the ZN-His coordination. Any help is highly appreciated. Thanks SDY Date: Thu, 8 Nov 2012 14:36:59 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics. Watch CHI2 :-) Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote: Then we agree. I got confused because you mentionedspace group and not point group in your phrase about PHASER and MOLREP and was afraid others might have gotten confused as well. Also, in case of twinning or almost crystallographic non-crystallographic symmetry, determining the point group on the basis of processing statistics alone can be inconclusive or even misleading. If I recall correctly, there has recently been a thread about this in the bulletin board. Herman 74_refmac5_1.log
Re: [ccp4bb] low-resolution and zinc
I am using Refmac_5.7.0029 in CCP4ThanksSDY Date: Fri, 9 Nov 2012 20:35:48 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK What program do you use for refinement?FF Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 9, 2012, at 20:09 , SD Y ccp4...@hotmail.com wrote:Dear All, Thanks for all the suggestions on low resolution, SG and Zn and I learned a lot. I am not done yet. I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger Rowlett. Also an excellent protocol is available at http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol seems to be very straight forward. I am not getting the result I wanted. 1. When ever I generate cif file, its not writing any for ZN-SG links at all. 2. After refmac refinement it only draw in LINK for His-ZN. Please see the imageshttps://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.pnghttps://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png 3. I get this His-Zn link without using .cif file, so what stage do I use this .cif file? 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing LINK in PDB. I only see LINKR ZNZN H 1 SG CYS A 83ZN-CYS I dont know what is missing, I also attached the log file which generated the ZN-His coordination. Any help is highly appreciated. ThanksSDY Date: Thu, 8 Nov 2012 14:36:59 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics.Watch CHI2 :-) Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com wrote:Then we agree. I got confused because you mentionedspace group and not point group in your phrase about PHASER and MOLREP and was afraid others might have gotten confused as well. Also, in case of twinning or almost crystallographic non-crystallographic symmetry, determining the point group on the basis of processing statistics alone can be inconclusive or even misleading. If I recall correctly, there has recently been a thread about this in the bulletin board.Herman 74_refmac5_1.log
[ccp4bb] side chain density
Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] side chain density
Hi, There has been quite a lot of discussion about this and I think different opinions exist. I would try to keep the side chains, if there is some evidence where they are. Otherwise I would just delete atoms and I would not mutate them to alanine. http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20437.html Best regards, Lari __ Lari Lehtiö, PhD, Adjunct Professor Biocenter Oulu Department of Biochemistry P.O.Box 3000 FIN-90014 University of Oulu Finland http://cc.oulu.fi/~llehtio/ _ Quoting Faisal Tarique faisaltari...@gmail.com: Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] side chain density
On Friday, 09 November 2012, Faisal Tarique wrote: Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such. Mutating to alanine is not an option. They are not alanine. If nothing else, when you get to the point of depositing your structure in the PDB it will fail validation checks because the sequence is not correct at those points. But if you mean should you delete sidechain atoms beyond CB, that is another question. That is a legitimate option. I suggest trying that and then looking in difference density maps to see if any density shows up to guide placement of the sidechain. Ethan .The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Assemble Protein-DNA complex
It is not very surprising that the affinity gets higher with lower salt, right? Why dont you measure it under _physiological_ salt concentration? (or i assume maybe you did?) and of course its not as high affinity due to screening (but physiological conditions) of the electrostatic interactions. If the complex doesnt stay together in ca. 150 mM salt (e.g in TBS) in gel filtration (which you dont say if you tried) then why not just try mixing it in the drop and screen, this is what you would do if you cant purify the complex. and try different rations. There are texts on preparation of DNA complexes in case you dont have advice for it. Tommi On Nov 9, 2012, at 6:50 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei Huang, if you are lucky you can form the complex as crystal in the drop (I assume you want to crystallise the protein-DNA complex): set up drops at high salt concentration (as low as possible to keep the protein in solution at reasonable concentration) in the presence of DNA. Prepare the buffer the same way as the protein buffer, but with reduced salt concentration (btw, you can also try divalent ions, eg. MgCl2). This way water evaporates into the drop, diluting the salt concentration. - From your discription the solubility of the protein drops faster than its concentration, hence it should precipitate. And, as I said, if you are lucky, it does so as a crystal in complex with the DNA. Best, Tim On 11/09/2012 05:14 PM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6 nvrZMfMmeekAauJ26jy6jbE= =nbV+ -END PGP SIGNATURE- Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
Re: [ccp4bb] usefulness of cacodylate?
On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? [...] (We're being subjected to a safety review.) I know you are in the UK but this wouldn't have anything to do with this: http://bostonglobe.com/lifestyle/health-wellness/2012/11/05/what-telling-patients-about-arsenic-and-rice/8VkGh3K3TtXEaucIzPEHvJ/story.html would it...? -Bryan
Re: [ccp4bb] side chain density
Dear Faisal, You definitely do not mutate to alanine as that would imply for the future user of your pdb file that it is a mutant. Some people feel they have to keep the side chain but put the occupancies at zero. I think this is a bad practice and strongly oppose to it as for the future user of your deposited pdb file, who often is not a crystallographer, you suggest a specific conformation for your side chain that may be interpreted in terms of biology, while in reality it addopts a huge, disordered ensemble of conformations. Personally I am of the opinion that you should simply remove the side chain atoms (but keep the residue name). And that is the same as what you do with a whole loop that is disordered. I think it is lso the most common practice in deposited structures. Remy Loris Vrije Universiteit Brussel On 09/11/12 20:22, Faisal Tarique wrote: Dear all i have solved a structure ( at 2A resolution) whose Rwork and Rfree is 22 and 25 respectively..the Ramachandran plot shows 90% of the residues in the most favorable region and with 6 residues in generously allowed and no residues in disallowed region. But in some areas i can see density missing for side chains ( in loop regions )..i have question do i need to mutate them to alanine or leave them as such..The density fit analysis in COOT ( traffic light) showing those regions with side chain as red.. thanx in advance Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Assemble Protein-DNA complex
On 11/09/12 15:43, Tommi Kajander wrote: It is not very surprising that the affinity gets higher with lower salt, right? Not at all. I wanted to ask if their assay could distinguish between specific binding and nonspecific binding, but I decided not to sidetrack the discussion. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] low-resolution and zinc
Are you trying to have Coot display the Zn-ligand bonds? That's a different issue from having refmac recognize and use the metal-ligand restraints in refinement. (I don't bother to have Coot do this.) Looking at your environment distances, it looks like refmac has done a restrained refinement, as your Zn-ligand bond distances look right. You can tell in the refmac log file if you get a message describing that a Zn-ligand distance was found and it is not listed as not be used. (If you get this latter message, you have the wrong link usage setting chosen in the CCP4i GUI.) I just had an undergraduate student do the procedure yesterday to do a restrained refinement for a ZnCys2HisAsp environment. Refmac picks up the Zn-Cys link in the library without any intervention, and the Zn-His and Zn-Asp bonds have to be taken care of in the refmac-generated .cif file. All the links show up in the LINKR records. There may be a more elegant way to do this but it works. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 11/9/2012 1:09 PM, SD Y wrote: Dear All, Thanks for all the suggestions on low resolution, SG and Zn and I learned a lot. I am not done yet. I am trying model co-ordinated ZN+2. I got lot of help from Prof. Roger Rowlett. Also an excellent protocol is available at http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement. Protocol seems to be very straight forward. I am not getting the result I wanted. 1. When ever I generate cif file, its not writing any for ZN-SG links at all. 2. After refmac refinement it only draw in LINK for His-ZN. Please see the images https://www.dropbox.com/s/7sl01pcdmxxcu2z/ZN-cpoordination-1.png https://www.dropbox.com/s/cxlp2m2stbple02/ZN-cpoordination-2.png 3. I get this His-Zn link without using .cif file, so what stage do I use this .cif file? 4. Though cysteines were placed around 2.2 - 2.5 A from ZN, its not writing LINK in PDB. I only see LINKR ZNZN H 1 SG CYS A 83ZN-CYS I dont know what is missing, I also attached the log file which generated the ZN-His coordination. Any help is highly appreciated. Thanks SDY Date: Thu, 8 Nov 2012 14:36:59 +0200 From: mbfro...@post.tau.ac.il Subject: Re: [ccp4bb] low-resolution and zinc To: CCP4BB@JISCMAIL.AC.UK The experiment should be very problematic if I can't determine point group on the base of the symmetry merging statistics. Watch CHI2 :-) Dr Felix Frolow Professor of Structural Biology and Biotechnology, Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il mailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 8, 2012, at 14:29 , herman.schreu...@sanofi.com mailto:herman.schreu...@sanofi.com wrote: Then we agree. I got confused because you mentionedspace group and not point group in your phrase about PHASER and MOLREP and was afraid others might have gotten confused as well. Also, in case of twinning or almost crystallographic non-crystallographic symmetry, determining the point group on the basis of processing statistics alone can be inconclusive or even misleading. If I recall correctly, there has recently been a thread about this in the bulletin board. Herman
