Re: [ccp4bb] freezing crystals grown in isopropanol condition
Chris Isopropanol is said a very good precipitant but it's unpopular because it's so difficult to harvest crystals However there's a very convenient way to grow and harvest crystals in isopropanol using Vapor Batch plates. Basically you set up microbatch-under-oil drops containing all the ingredients except for the isopropanol, and cover with Al's Oils (silicone mixed with paraffin oil). Then you put e.g. in your case 25% isopropanol in the reservoir around the outside of the Vapor Batch plate. Over a few hours the isopropanol will diffuse through the Al's Oils into the drops. The great advantage is that the oil also becomes saturated with isopropanol (they are only partially miscible in fact) so that the oil acts as a barrier when you're harvesting crystals. You can usually convert vapor diffusion conditions to microbatch in a single 24-well experiment where you vary protein against precipitant. You don't need to remove the oil to collect data. This is all described in Lesley Haire's winning entry to a competition we organized a few years ago: http://www.douglas.co.uk/winner1.htm See also Mortuza et al. High-resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431 (2004), pp 481-485. The Vapor Batch plate can be seen at http://www.douglas.co.uk/vb.htm If you or anyone on the b.b. would like to try some samples of Vapor Batch plates just let me know Best wishes Patrick On Fri, Apr 9, 2010 at 9:53 AM, Chris Meier crystallogra...@christophmeier.com wrote: Dear all, I have a protein which crystallizes in 25% isopropanol, at pH4.5. Does anyone have experience freezing crystals grown in such a condition? What cryoprotectants should I try? Can isopropanol itself act as a cryoprotectant? Any suggestions on how to deal with isopropanol evaporation during mounting? Many thanks and best wishes, Chris -- patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] phosphate v sulfate
Dear colleagues, I would like to poll the community on the prevailing practice of distinguishing between phosphate and sulfate in their structures. Suppose all of the following apply: 1) a well contoured tetrahedral density in your model-phased 2Fo-Fc map in the active site of your kinase or GTPase protein. 2) a significant, say 5*sigma, coincident peak in your model-phased anomalous difference fourier map from data collected at a Cu rotating anode source. 3) The crystal grew in 2M ammonium sulfate. Please post your answers to the list if you feel this question is of general interest.* Thank you for your input. Wolfram Tempel *If it is not, I apologize to everyone for wasting their valuable time.
Re: [ccp4bb] phosphate v sulfate
Hello, Thanks for this interesting poll! I have had similar doubt couple of times in the recent past. I think in the absence of anomalous signal for confirming presence of phosphate or sulphate, I would prefer to assign the electron density to the one which is present in higher concentration in the crystallization drop. Just on the basis of electron density, co-ordination geometry and functional groups attached to the tetrahedral density, I assume it is very difficult to say if it is phosphate or sulphate. In your case, I would go for SO4 instead of PO4. I would also like to know other people's experiences in such cases. Regards Simanshu On Sat, Apr 10, 2010 at 10:49 AM, wtempel wtem...@gmail.com wrote: Dear colleagues, I would like to poll the community on the prevailing practice of distinguishing between phosphate and sulfate in their structures. Suppose all of the following apply: 1) a well contoured tetrahedral density in your model-phased 2Fo-Fc map in the active site of your kinase or GTPase protein. 2) a significant, say 5*sigma, coincident peak in your model-phased anomalous difference fourier map from data collected at a Cu rotating anode source. 3) The crystal grew in 2M ammonium sulfate. Please post your answers to the list if you feel this question is of general interest.* Thank you for your input. Wolfram Tempel *If it is not, I apologize to everyone for wasting their valuable time. -- Dhirendra K Simanshu Research Scholar Memorial Sloan-Kettering Cancer Center New York, NY, USA
Re: [ccp4bb] phosphate v sulfate
One might also check the pH of your crystal and the number of hydrogen bond donors from the putative sulfate (zero?) or the putative phosphate (non-zero). I'm alluding to the structures of the sulfate-binding protein and the phosphate-binding protein from Quiocho's group. Jim _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of wtempel Sent: Saturday, April 10, 2010 9:49 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] phosphate v sulfate Dear colleagues, I would like to poll the community on the prevailing practice of distinguishing between phosphate and sulfate in their structures. Suppose all of the following apply: 1) a well contoured tetrahedral density in your model-phased 2Fo-Fc map in the active site of your kinase or GTPase protein. 2) a significant, say 5*sigma, coincident peak in your model-phased anomalous difference fourier map from data collected at a Cu rotating anode source. 3) The crystal grew in 2M ammonium sulfate. Please post your answers to the list if you feel this question is of general interest.* Thank you for your input. Wolfram Tempel *If it is not, I apologize to everyone for wasting their valuable time.
Re: [ccp4bb] Does the substrate has access to the active site?
Hi, I am assuming the enzyme is not active, or by substrate you do not mean the actual substrate, may be an analogue. The substrate might be converted into product and the leave through the channel and you will not find anything bound to it. But I think you have taken care of that. On Fri, Apr 9, 2010 at 7:00 AM, Ed Pozharski epozh...@umaryland.edu wrote: It is also possible that mother liquor prevents binding (although often in such cases you would see some precipitant component in the active site. I would generally bet on need for conformational change. And you expect to see the product complex, right? Ed. On Fri, 2010-04-09 at 11:15 +0200, Paul Lindblom wrote: Dear Bulletin Board, I am trying to soak substrate into crystals of an enzyme, but so far I can't see the substrate in the structure. Does anyone knows a program to ensure that the entrance to the central cavity is accessibly? I mean based on the whole crystal. I already checked the crystal packing manually and it seems that the way is free more or less, but I find it hard to interpret. Thanks in advance, P. -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] problems with molprobity in coot
Rongjin, It does look like you have the same problem I did. I was never totally able to figure it out completely. First of all, it was only a problem with some PDB files and not others. Modifying generic_objects.py (i.e., change the line if (reduce_status): to if (True): ) as suggested by Paul Emsley and Bernhard Lohkamp did seem to fix the problem for some PDB files, but for others, I would then encounter a second error that I couldn't get past. I don't remember the details of the second error, and I'm not on that computer, so I can't get any more details right now. I think that ultimately I reprocessed my data (for an unrelated reason) and the problem went away, but it was a few months ago and I can't remember all the details. Sorry I could not be of more help! -Tom On Wed, Apr 7, 2010 at 11:06 PM, Rongjin Guan rjg...@gmail.com wrote: Hi Thomas I have a similar problem and wonder if you can help. I just installed Wincoot 0.6.1 and reduce/probe as well, and tested with several PDB files for probe/clash validation. For some PDBs it works perfectly but for another two, it did not work and I have the following message: Found 0 hydrogens (0 hets) Standardized 0 hydrogens (0 hets) Added 3946 hydrogens (0 hets) Removed 0 hydrogens (0 hets) Adjusted 113 group(s) If you publish work which uses reduce, please cite: Word, et. al. (1999) J. Mol. Biol. 285, 1735-1747. For more information see http://kinemage.biochem.duke.edu BL WARNING:: reduce didnt run ok, so stop here! run_generic_script (probe, 0) I saw you had this message before; did you figure out what happened? Thanks Rongjin Guan = = = = = = = = On 2009-10-12 12:37:34 You wrote = = = = = = = = Bernhard, I have tried this using several versions of coot. I tried it first in the coot that came packaged with ccp4 (Coot 0.5.2). I then tried WinCoot-0.6-pre-1-revision-2411, which is one of the later revisions of 0.6-pre, just to see if a more recent version would work. As far as I can tell, I am using the correct versions of probe and reduce. I am attaching a file with the relevant portion of the output (starting with when I run probe clashes, up until it fails). Thanks for your help! -Tom On Mon, Oct 12, 2009 at 3:21 AM, Bernhard C. Lohkamp bernh...@chem.gl.ac.uk wrote: Thomas, I am trying to use molprobity to check a structure in coot (platform is 64-bit vista). I followed the instructions found here to set up molprobity: http://www.ysbl.york.ac.uk/~lohkamp/coot/wincoot-faq.htmlhttp://www.ysbl.york.ac.uk/%7Elohkamp/coot/wincoot-faq.html . From the logs, it seems like reduce runs OK (it says that it Added 6076 hydrogens). However, at the very end, I get the message BL WARNING:: reduce didnt run ok, so stop here! It seems like probe never runs. Does anyone know how to solve this? Maybe you can give me some more information. Which version of WinCoot are you running and which versions of probe and reduce are you using? Make sure you use the right version of probe (2.12.something) and reduce (3.13.something). Note to self: update the website with respect to versions. If have the 'right' versions please sent me the console output. Hopefully that will give more information. B
