CCP4BB automatic digest system <[email protected]> wrote:
>There are 5 messages totaling 519 lines in this issue. > >Topics of the day: > > 1. freezing crystals grown in isopropanol condition > 2. phosphate v sulfate (3) > 3. Does the substrate has access to the active site? > >---------------------------------------------------------------------- > >Date: Sat, 10 Apr 2010 11:09:08 +0100 >From: Patrick Shaw Stewart <[email protected]> >Subject: Re: 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 < >[email protected]> 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 >> >> >> > > > >-- >[email protected] Douglas Instruments Ltd. >DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK >Directors: Peter Baldock, Patrick Shaw Stewart > >http://www.douglas.co.uk >Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 >Regd. England 2177994, VAT Reg. GB 480 7371 36 > >------------------------------ > >Date: Sat, 10 Apr 2010 10:49:08 -0400 >From: wtempel <[email protected]> >Subject: 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. > >------------------------------ > >Date: Sat, 10 Apr 2010 12:11:16 -0400 >From: Dhirendra K Simanshu <[email protected]> >Subject: Re: 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 <[email protected]> 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 > >------------------------------ > >Date: Sat, 10 Apr 2010 13:21:52 -0500 >From: Jim Pflugrath <[email protected]> >Subject: Re: 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:[email protected]] On Behalf Of >wtempel >Sent: Saturday, April 10, 2010 9:49 AM >To: [email protected] >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. > >------------------------------ > >Date: Sat, 10 Apr 2010 12:43:10 -0700 >From: xaravich ivan <[email protected]> >Subject: Re: 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 <[email protected]> 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 / >> > >------------------------------ > >End of CCP4BB Digest - 9 Apr 2010 to 10 Apr 2010 (#2010-97) >***********************************************************
