CCP4BB automatic digest system <[log in to unmask]> 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 <[log in to unmask]>
>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 <
>[log in to unmask]> 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
>>
>>
>>
>
>
>
>-- 
>[log in to unmask]    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 <[log in to unmask]>
>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 <[log in to unmask]>
>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 <[log in to unmask]> 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 <[log in to unmask]>
>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:[log in to unmask]] On Behalf Of
>wtempel
>Sent: Saturday, April 10, 2010 9:49 AM
>To: [log in to unmask]
>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 <[log in to unmask]>
>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 <[log in to unmask]> 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)
>***********************************************************