Re: [ccp4bb] Crystals grow at the bottom of the tube.

[Patrick Shaw Stewart]

Hi Lingyuan

I would certainly make a seed stock and use it with RANDOM screens - this
will (usually) allow you to pick up new conditions and control the number
of crystals per drop.

However you say your crystals melt easily when you harvest them.  I don't
know how David stabilized his seedstock (if he did) but you may be able to
stabilize yours with a precipitant.

In all the cases that we looked at we found that if you soaked uncrushed
crystals in a precipitant (or cocktail) and the crystals were unchanged
after 24 hours, then the precipitant/cocktail could be used to make a
seedstock.

In practice 100% PEG 600 worked for 5 of the 6 model proteins that we
looked at.  The remaining seedstock worked with seed crystals suspended in
4M amm. sulfate.

I hope it works for you.

Best wishes, Patrick


___


Ref for stabilizing seed stocks with various precipitants:

Shaw Stewart, P.D., Kolek, S.A., Briggs, R.A., Chayen, N.E. and Baldock,
P.F., 2011. Random microseeding: a theoretical and practical exploration of
seed stability and seeding techniques for successful protein
crystallization.*Crystal Growth & Design*, *11*(8), pp.3432-3441.



Ref for random microseeding:

D'Arcy A, Villard F, Marsh M. An automated microseed matrix-screening
method for protein crystallization. Acta Crystallographica Section D:
Biological Crystallography. 2007 Apr 1;63(4):550-4.





On 19 October 2016 at 15:20, Hargreaves, David <
david.hargrea...@astrazeneca.com> wrote:

> I have been presented with crystals grown in nmr tubes on two separate
> occasions. Both diffracted very well and thankfully, neither required a
> magnetic field for optimisation. On the first occasion I did use the
> initial sample as a seed stock.
>
>
>
> Best wishes,
>
>
>
> David
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *
> kiki
> *Sent:* 18 October 2016 11:42
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] Crystals grow at the bottom of the tube.
>
>
>
> Dear all users,
>
>
>
>  I got my crystals at the bottom of the tube on ice just before I started
> to screen crystals. Those crystals' shapes were good but easy to melt when
> I harvested them. I shot these crystals. They only diffracted to 7A to 8A.
> Has anyone ever met this situation before? How to improve?
>
>
>
> Thanks,
>
> Lingyuan
> --
>
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> www.astrazeneca.com
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Re: [ccp4bb] Crystals grow at the bottom of the tube.

[Hargreaves, David]

I have been presented with crystals grown in nmr tubes on two separate 
occasions. Both diffracted very well and thankfully, neither required a 
magnetic field for optimisation. On the first occasion I did use the initial 
sample as a seed stock.

Best wishes,

David

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of kiki
Sent: 18 October 2016 11:42
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystals grow at the bottom of the tube.

Dear all users,

 I got my crystals at the bottom of the tube on ice just before I started to 
screen crystals. Those crystals' shapes were good but easy to melt when I 
harvested them. I shot these crystals. They only diffracted to 7A to 8A. Has 
anyone ever met this situation before? How to improve?

Thanks,
Lingyuan


AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number:03674842 and its registered office at 1 Francis Crick Avenue, 
Cambridge Biomedical Campus, Cambridge, CB2 0AA.

This e-mail and its attachments are intended for the above named recipient only 
and may contain confidential and privileged information. If they have come to 
you in error, you must not copy or show them to anyone; instead, please reply 
to this e-mail, highlighting the error to the sender and then immediately 
delete the message. For information about how AstraZeneca UK Limited and its 
affiliates may process information, personal data and monitor communications, 
please see our privacy notice at 
www.astrazeneca.com

Re: [ccp4bb] Crystals grow at the bottom of the tube.

[mesters]

Yes, it is many years ago, a large protein batch (50 ml or so) stored in 
the cold room that produced an impressive layer of crystals at the 
bottom of the flask.
One student at that time tested many crystals and found only a few that 
diffracted to atomic (!) resolution.


Crystallization is a usefull methodology for protein purification! What 
I would do is to harvest these crystals (often more pure protein than in 
the stock solution), dissolve them and try to grow new crystals as 
proposed by Artem.


Good luck!

Jeroen


Am 18.10.16 um 12:41 schrieb kiki:

Dear all users,

 I got my crystals at the bottom of the tube on ice just before I 
started to screen crystals. Those crystals' shapes were good but easy 
to melt when I harvested them. I shot these crystals. They only 
diffracted to 7A to 8A. Has anyone ever met this situation before? How 
to improve?


Thanks,
Lingyuan


--
Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/ 



Institute of Biochemistry, University of Lübeck
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http://www.iobcr.org 


--
If you can look into the seeds of time and tell which grain will grow 
and which will not, speak then to me who neither beg nor fear 
(Shakespeare's Macbeth, Act I, Scene 3)

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Re: [ccp4bb] Crystals grow at the bottom of the tube.

[Artem Evdokimov]

Hi Lingyuan,

1) you're very lucky (but you already know that)
2) crystals grown quickly in bulk rarely diffract well. I would follow up
by a conventional droplet crystallization against a concentrating agent -
in the cold - such that your protein gradually concentrates from [X] to 2-3
times [X] and that should presumably give you crystals. Another option is
temperature-driven crystallization (if this is what is happening) in which
case a simple setup consists of a sitting or hanging drop plate inside a
closed styrofoam box that you set up at room temperature, then bring the
box (already closed) into a cold room - the box adds a considerable delay
factor. If you want to extend the cooling process even more you could place
a few Blue Ice packs in the box (again, at room temperature).

Artem

www.harkerbio.com
"protein crystals for breakfast, lunch, AND dinner"


- Cosmic Cats approve of this message

On Tue, Oct 18, 2016 at 6:41 AM, kiki  wrote:

> Dear all users,
>
>  I got my crystals at the bottom of the tube on ice just before I started
> to screen crystals. Those crystals' shapes were good but easy to melt when
> I harvested them. I shot these crystals. They only diffracted to 7A to 8A.
> Has anyone ever met this situation before? How to improve?
>
> Thanks,
> Lingyuan
>

[ccp4bb] Crystals grow at the bottom of the tube.

[kiki]

Dear all users,


 I got my crystals at the bottom of the tube on ice just before I started to 
screen crystals. Those crystals' shapes were good but easy to melt when I 
harvested them. I shot these crystals. They only diffracted to 7A to 8A. Has 
anyone ever met this situation before? How to improve?


Thanks,
Lingyuan