Dear Patrick,
You along with others had made some suggestions last time. May be its a good
time to update.
With classical screening, I got a crystal like appearances/shower with HEPES
7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and with
different conc of Lithium I could only get very very thin needles which shower
and difficult to pick even with 0.05 loop. changing conc of protein, salt,
adding oil on well, changing drop ratio, adding 5% PEGs, glycerol, ethylene
glycol, didnt reduce shower.
I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS screened
with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) 100nl+100nL+50nL
seeds using Phoenix.
Got several nice hits which very good size individual crystals in conditions
with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. When I optimized
them I got beautiful crystals and tried at ALS 5.0.3 but no spots. I tried
picking crystals from 30 min to 4 hrs to 4 days after plates were set and there
was no luck. Crystals started to appear from 20 min onwards and keep growing in
next couple of hours. I thought of trying dehydration but they were already in
dehydrating conditions them selves. I wanted to ask if anyone ever failed with
MMS but thought not waste others time on this. Cross seeding to full length
protein and Se met protein also gave beautiful crystal but again no
diffraction. I have not checked SeMet as they were bit small.
I am still open to ideas if you have any thing on seeding. I did try in
microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul
seeds, same as sitting drop which gave me very good looking crystals) but I
didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but it
didn't give me anything.
Currently, I am making entropy mutations, new constructs of different lengths
to solve the above problem.
I still want to improve this condition with needles, because I collected a 4.5A
data on one of the needle (just one). I need phases. I have sent some Iodide
soaks to synchotron (yet to collect data) but manipulating these crystal drops
with more than 100 tiny needles with a tough membrane on it has been
frustrating as I end up loosing several drops to just to fish out 1-2 needles.
I am ready to try if there any trick left.
Thanks for lots and lots of help.
Regards,Rajesh
Date: Fri, 11 May 2012 18:09:54 +0100
Subject: Re: [ccp4bb] zinc with HEPES
From: patr...@douglas.co.uk
To: ccp4...@hotmail.com
Rajesh
How did you do the MMS? By hand or with a robot, and what screens did you use?
and why did you change to HEPES out of interest?
Patrick
On 11 May 2012 18:05, Rajesh Kumar <ccp4...@hotmail.com> wrote:
The rationale was to see if Zn could make differences in crystal morphology.
This is because the protein has CxxC and CxxH similar to a zinc finger
motif.All my efforts, additive screening, MMS, streaking, micro batch, hanging
drop, changing drop ratio, drop shape, did not help me to either increase
thickness or change the shape of very very thin needle crystals.
Yes, I will try very less, 50uM.Thanks for helping me to understand.Rajesh
> Date: Fri, 11 May 2012 12:53:53 -0400
> Subject: Re: [ccp4bb] zinc with HEPES
> From: liehy...@gmail.com
> To: ccp4...@hotmail.com
>
> Rajesh,
> 10mM zinc seems a bit too high. I normally used it at <50uM conc.
> ray
>
> On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar <ccp4...@hotmail.com> wrote:
> > Dear All,
> >
> > This question sounds simple but I dont know the answer.
> > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4
> > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn
> > acetate the effect is same.
> > I dont know why this Zn in not compatible with HEPES.
> > Could you please tell me why is this?
> > I appreciate your help.
> >
> > Thanks
> > Rajesh
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