Re: [ccp4bb] Problems installing CCP4 6.2 on Mac OS X 10.7.3
1, Check your directory permission. Make sure you have the read and execute permission for that directory (sometimes like this* drwxr-xr-x*). 2, Try to install CCP4 package by *fink*. There's already a stable version for Lion. (http://pdb.finkproject.org/pdb/package.php/ccp4) On Tue, May 22, 2012 at 12:44 AM, Terry Lang te...@lego.berkeley.eduwrote: Hey Everyone, I am having some problems getting CCP4 up and running on a laptop with Mac OS X 10.7.3. I have downloaded the .dmg and run the installation with no errors. However, when I try to set up my Project directory, I run into some problems. It seemingly won't let me access my directory tree. When I click on Users, for example, the Browse window does not enter the directory and frequently just closes without an error message. Any ideas? Thanks, Terry -- Paula Therese Lang Postdoctoral Scholar Alber Lab UC Berkeley/QB3 -- Xiaoguang Xue, PhD student Utrecht University Crystal Structural Chemistry Padualaan 8. Room N807 3584 CH Utrecht The Netherlands Tel. +31-30-253-2383
Re: [ccp4bb] Serine
lovely density - i would like to know what % of series have multiple conformations - it would need a survey I guess of PDB depositions, not just coordinates, but checking maps.. Eleanor On 21 May 2012 22:21, Uma Ratu rosiso2...@gmail.com wrote: Thank you All for you inputs. Uma On Mon, May 21, 2012 at 5:06 PM, Van Den Berg, Bert lambertus.vandenb...@umassmed.edu wrote: Yes, as Jacob says, alternative conformation of the serine. Quite common. Bert -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Uma Ratu [rosiso2...@gmail.com] *Sent:* Monday, May 21, 2012 4:57 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Serine Dear All: Some of serine residues in my model have extra positive Fo-Fc density at the edge of side chain. Some don't have. It is not like from phosphates. I am wondered what is the cause for these extra density. Could these serines be post-translational modified? I have the images attached. P289ser-0512-1 does not have the extra green, where P140ser-0512-1 has. Thank you for you advice and comment Uma
Re: [ccp4bb] Serine
Dear Uma 1. The protein sequence given in the databases, do from time to time have errors, particularly if you are working with old proteins (when the sequencing was done a long time ago). What you observe could also be a threonine or even a valine. You should check whether homologous protein also have a serine on that position. I Normally do not assign double conformation unless you have fairly high resolution data, it is a bit hard to judge what the resolution is here. Often a double conformation is accompanied with negative difference density on top of the assigned atom, if you lower the sigma level a little, you often see it. 2. It is also common to have local frameshift errors in loop regions, even in reasonable high resolution data. I think you should double check the region to make sure you are not facing this problem, it is surprisingly difficult to detect sometimes, but you will know when you got the right. Cheers Preben On 5/21/12 10:57 PM, Uma Ratu wrote: Dear All: Some of serine residues in my model have extra positive Fo-Fc density at the edge of side chain. Some don't have. It is not like from phosphates. I am wondered what is the cause for these extra density. Could these serines be post-translational modified? I have the images attached. P289ser-0512-1 does not have the extra green, where P140ser-0512-1 has. Thank you for you advice and comment Uma -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] Serine
3. Of course if you are working on recombinant protein, you should double check the sequencing results from the cloning Preben On 5/21/12 11:21 PM, Uma Ratu wrote: Thank you All for you inputs. Uma On Mon, May 21, 2012 at 5:06 PM, Van Den Berg, Bert lambertus.vandenb...@umassmed.edu mailto:lambertus.vandenb...@umassmed.edu wrote: Yes, as Jacob says, alternative conformation of the serine. Quite common. Bert *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Uma Ratu [rosiso2...@gmail.com mailto:rosiso2...@gmail.com] *Sent:* Monday, May 21, 2012 4:57 PM *To:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Serine Dear All: Some of serine residues in my model have extra positive Fo-Fc density at the edge of side chain. Some don't have. It is not like from phosphates. I am wondered what is the cause for these extra density. Could these serines be post-translational modified? I have the images attached. P289ser-0512-1 does not have the extra green, where P140ser-0512-1 has. Thank you for you advice and comment Uma -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
[ccp4bb] Fwd: Crystallographic Software Fayre (ECM27, Bergen)
Hi folks The list of microsymposia at ECM27 is now available, but the list of speakers has not yet been finalised. Bear in mind that earlybird registration closes on 31st May, when the cost jumps by 750 NOK (~€100) for full participants, 550NOK (~€72) for students. The meeting website is http://ecm27.ecanews.org/ On 11 May 2012, at 10:36, Harry Powell wrote: Hi folks Any of you who are considering attending ECM27 in Bergen may be interested in some of the sessions that have been arranged at this Software Fayre. The full list of Microsymposia at ECM27 should be available in the next couple of days From: Martin Lutz m.l...@uu.nl Date: 10 May 2012 13:48:11 GMT+01:00 To: undisclosed-recipients:; Subject: Crystallographic Software Fayre (ECM27, Bergen) Dear all, thanks to the local organizers, there will be a Crystallographic Software Fayre at the ECM27 meeting in Bergen (Norway). Authors of of academic and/or open-source software can present their new developments. The presentations should have a tutorial character with one or more practical examples. The available time slots can be seen on the website: http://www.cryst.chem.uu.nl/lutz/software_fayre.html (This website will be updated regularly) If you are interested, please send an e-mail with your name and a preliminary title of the presentation to Martin Lutz, m.l...@uu.nl. The time slots will be filled according to the first-come first-served principle. For information about the ECM27 congress (August 6-11, 2012), see: http://ecm27.ecanews.org With kind regards, Martin (Please forward this e-mail to anyone, who might be interested.) -- Martin Lutz Crystal and Structural Chemistry Bijvoet Center for Biomolecular Research Faculty of Science Utrecht University Padualaan 8 3584 CH Utrecht The Netherlands Tel. [+31] 030-2533902 Fax [+31] 030-2533940 Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH
Re: [ccp4bb] color for metal ions
thanks everybody. On Mon, May 21, 2012 at 1:51 PM, mjvdwo...@netscape.net wrote: There is a really nice web site that shows how colors are perceived by various color-blind readers. One of the journals I recently published in recommends it for consideration. If you are interested, have a look. The web site is made especially for people who publish scientific articles with color illustrations. It is not hard to be considerate. http://jfly.iam.u-tokyo.ac.jp/html/color_blind/ Mark -Original Message- From: Artem Evdokimov artem.evdoki...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Sun, May 20, 2012 2:54 pm Subject: Re: [ccp4bb] color for metal ions As long as you are considerate of the needs of colorblind people, I would vote that anything goes. Artem On May 20, 2012 3:17 PM, sujata halder halder.suj...@gmail.com wrote: Hi all, I was wondering if there is a rule for coloring metal ions a specific color. I am using pymol and was not sure if I have to use a specific color for calcium or magnesium ions for publication figures. Thanks, Sujata
[ccp4bb] Purify protein-DNA complex
Dear CCP4ers, I am working on purifying a protein-DNA complex for structural and biochemical studies. So far, I can readily make protein 95% pure in high salt buffer. However, I have some problems in assembling the protein-DNA complex. 1) My protein precipitates at low salt buffer. I think this is the source of all my problems. 2) So I tried DNA binding at high salt buffer. Unfortunately, my protein precipitates after adding DNA (1.2:1 ratio, and DNA duplex was annealed by heat and slowly cool) and incubating at RT for 10 mins. 3) Then I found some posts here and tried DNA binding at very low concentration ( 0.2 mg/mL protein) at high salt buffer, which seems to alleviate the precipitation. And I concentrated protein and DNA mixture, and loaded it on to FPLC to further purify protein-DNA complex. However, I didn't observe the shift of my protein peak upon DNA binding (my protein is a 76 kDa dimer, and DNA duplex is 11 kDa) and I can see a high DNA peak at the position of 17kDA protein standard peak. Therefore, my protein doesn't bind DNA at high salt as expected. 4) The next thing I tried is having DNA binding at low protein concentration while dialyzing in high salt buffer. And slowly titrated in low salt buffer (1mL/min) to a final concentration of 100 mM NaCl. However, this doesn't solve the problem of protein precipitation and I still lose majority of my protein resulting in the purity is 60%. BTW, the 5% impurity band looks like to be HSP (nearby 75kDa protein marker on an SDS-PAGE). But it seems that the HSP doesn't bind to my protein since my protein still forms dimer. I think it just co-elute with my protein because my last step purification--size exclusive column cannot separate them. If you have come across similar problems and found ways to solve the problems, could you share your experience with me? Thank you very much! I really appreciate your help. Best, -- Alex Huang Research Associate Institute for Cancer Research Xi'an Jiaotong University Xi'an, Shaanxi 710049 China
Re: [ccp4bb] Purify protein-DNA complex
Wei, I am not sure if I can solve your problem, but I can try to give insight. I am also working with a similar protein-DNA complex situation. I normally purify the protein by itself, and maintain the salt at 250 mM or greater. Addition of 1.5x DNA stabilizes this complex and I am able to dialyze the sample into 50 mM salt with no precipitation issues. In addition, I always check every piece of DNA using fluorescence anisotropy to make sure it actually binds. To comment on your points: 1) this is a common issue I think with most DNA binding proteins. Addition of DNA *usually* stabilizes the protein and allows for lower salt. 2) are you certain that your protein can bind DNA at high salt? usually high salt decreases the Kd since you are disrupting the electrostatic interactions made between the dna binding domain of your protein and the dna itself. Also, I would try incubating on ice instead of RT. In addition, what are the differences in the DNA buffer and protein buffer? Are there large pH differences? Perhaps your protein doesn't like basic solutions, which most DNA is normally annealed/resuspended in. 3) Since you are seeing a large DNA elution peak on your sizing column, this almost suggests to me that your DNA is not binding. This may seem like a dumb thing, but I would check and make sure that your protein can actually bind the DNA sequence you are trying, using something like FA/FP and a labelled oligo. 4) Again, this makes me think it isn't really binding the DNA, since your protein is crashing out. I think you could check this if you do a high-speed spin to pellet the protein precipitant and check it to see if the DNA is crashing out with it? Or check the supernatant to calculate the DNA concentration. These are just some suggestions I can think of off the top of my head, Bret On Tue, May 22, 2012 at 10:03 AM, Wei Huang xjt...@gmail.com wrote: Dear CCP4ers, I am working on purifying a protein-DNA complex for structural and biochemical studies. So far, I can readily make protein 95% pure in high salt buffer. However, I have some problems in assembling the protein-DNA complex. 1) My protein precipitates at low salt buffer. I think this is the source of all my problems. 2) So I tried DNA binding at high salt buffer. Unfortunately, my protein precipitates after adding DNA (1.2:1 ratio, and DNA duplex was annealed by heat and slowly cool) and incubating at RT for 10 mins. 3) Then I found some posts here and tried DNA binding at very low concentration ( 0.2 mg/mL protein) at high salt buffer, which seems to alleviate the precipitation. And I concentrated protein and DNA mixture, and loaded it on to FPLC to further purify protein-DNA complex. However, I didn't observe the shift of my protein peak upon DNA binding (my protein is a 76 kDa dimer, and DNA duplex is 11 kDa) and I can see a high DNA peak at the position of 17kDA protein standard peak. Therefore, my protein doesn't bind DNA at high salt as expected. 4) The next thing I tried is having DNA binding at low protein concentration while dialyzing in high salt buffer. And slowly titrated in low salt buffer (1mL/min) to a final concentration of 100 mM NaCl. However, this doesn't solve the problem of protein precipitation and I still lose majority of my protein resulting in the purity is 60%. BTW, the 5% impurity band looks like to be HSP (nearby 75kDa protein marker on an SDS-PAGE). But it seems that the HSP doesn't bind to my protein since my protein still forms dimer. I think it just co-elute with my protein because my last step purification--size exclusive column cannot separate them. If you have come across similar problems and found ways to solve the problems, could you share your experience with me? Thank you very much! I really appreciate your help. Best, -- Alex Huang Research Associate Institute for Cancer Research Xi'an Jiaotong University Xi'an, Shaanxi 710049 China
[ccp4bb] postdoctoral position in Warsaw
The group of Dr. Marcin Nowotny at the International Institute of Molecular and Cell Biology (IIMCB) in Warsaw, Poland is seeking candidates for postdoctoral fellows. The fellows will work on protein complexes involved in DNA and RNA metabolism using protein crystallography and protein biochemistry (for examples of our previous work please see: Jaciuk M. et al. Nat. Struct. Mol. Biol. 18(2):191-7 and Rychlik M.P., et al. Mol. Cell 40(4):658-70). Further information about IIMCB can be found at: http://www.iimcb.gov.pl. The Institute has state-of-the-art equipment and facilities, including crystallization robots, an automated crystallization station and a microfocus home X-ray source. The candidates should hold a Ph. D. degree, must be motivated, well-organized and able to work independently as well as a part of the team. Experience with protein expression, purification and biochemical characterization is required. Knowledge and experience in protein crystallography will be an advantage. The candidates should send their detailed CV to mnowo...@iimcb.gov.plmailto:mnowo...@iimcb.gov.pl with reference contact information.
[ccp4bb] Calculating ED Maps from structure factor files with no sigma
Hello everyone, My apologies if this comes as basic, but I wanted to get the expert's take on whether or not the sigmaF values are required in the calculation of an electron density map. If I look at the standard ED equation, sigma's don't appear to be a requirement but all the scripts that I've looked at do require sigma values. I wanted to calculate the electron density for PDB id: 1HFS but the structure file only lists the Fo's, Fc's and Phases, but no sigmas. Would such structure factor file be considered incomplete? Thank you for your kind explanation. Francisco
Re: [ccp4bb] Calculating ED Maps from structure factor files with no sigma
Your understanding is correct, sigmaF values aren't required for calculating electron density. Many programs that calculate maps have an option to use the F/sigmaF ratio to threshold the amplitudes used in map calculation - which would require sigmaF. This isn't something I've seen used recently. The presence of sigF is also sometimes used as a proxy for confirming that the data is observed rather than calculated. Pete Francisco Hernandez-Guzman wrote: Hello everyone, My apologies if this comes as basic, but I wanted to get the expert’s take on whether or not the sigmaF values are required in the calculation of an electron density map. If I look at the standard ED equation, sigma’s don’t appear to be a requirement but all the scripts that I’ve looked at do require sigma values. I wanted to calculate the electron density for PDB id: 1HFS but the structure file only lists the Fo’s, Fc’s and Phases, but no sigmas. Would such structure factor file be considered incomplete? Thank you for your kind explanation. Francisco
