Re: [ccp4bb] salt or not?

[Patrick Shaw Stewart]

Careina,

One thing to try if other ideas don't work or are too difficult, is
covalently (therefore unambiguously) labelling a little of your protein
with a fluorescent dye.  If you add 20 nL of this to the drop *after the
crystals have grown*, protein crystals will light up, but salt crystals
will not.  Thermo make some very easy-to-use kits for labelling.  See
methods section of our paper *Cryst. Growth Des.*, 2011, *11* (8), pp
3432–3441.

Could you also label the DNA   . . .   ?

Hope it helps, best wishes, Patrick


On 15 April 2013 11:18, Careina Edgooms  wrote:

> Dear ccp4
>
> I have been performing trials on a protein DNA complex for a while now and
> have not seen any crystals form. Today I checked an old plate (over a month
> old) and I see 4 large crystals. *excitement* Three of them look tetragonal
> in shape (like a pyramid) and one of them looks hexagonal. I do not know if
> they are salt or protein. There is calcium chloride in the buffer. They
> feel quite soft to touch. They do not cause much birefringence. One of them
> does not seem to absorb much izit. It did go a bit blue but not entirely.
>
> How can I tell if this crystal is protein or not? Do you think its worth
> trying to see how it diffracts?
>
> Also, does Izit affect diffraction/ protein structures at all? Could I use
> a crystal with Izit in a diffraction experiment and ultimately to get the
> structure?
>
> Best
> Careina
>



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Re: [ccp4bb] salt or not?

[Ed. Pozharski]

Protein-DNA complex crystal with channels too small for the dye is *extremely* 
unlikely, imho.

 Original message 
From: Ulrike Demmer  
Date: 04/15/2013  8:48 AM  (GMT-05:00) 
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] salt or not? 
 
Dear Careina,

altough your crystals does't take up the Izit dye it sounds promising. The 
uptake of Izit depends on the solvent channels of the protein molecule - 
sometimes the dye just can't enter the molecule.
Concerning the Calciumchloride - if the concentration is not too high and 
without other ingredients which could cause less solube salt there is the 
possiblity that you have got protein crystals. I once had a condition whith 34 
% MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting  
crystals
To speak from my experience I think 1 month after setting up the trays the 
drops should not be dried out. If the wells are sealed properly new crytals can 
appear even after 1 year.

You should definately check the diffraction then you will know for sure.

Cheers,

Ulrike

Re: [ccp4bb] salt or not?

[James Holton]

I may be biased, but the only way to really be sure is to shoot them.

If you see no spots at all, be sure to do a wide "oscillation" (rotation 
during the exposure) shot as well.  It is not unlikely for a salt 
crystal to be oriented so that no relps are on the Ewald sphere, giving 
no spots.  But, if you sweep through 180 degrees during the exposure you 
will at least have a nice, pretty symmetric diffraction pattern to look 
at for a moment before the disappointment sets in.


-James Holton
MAD Scientist

On 4/15/2013 3:18 AM, Careina Edgooms wrote:

Dear ccp4

I have been performing trials on a protein DNA complex for a while now 
and have not seen any crystals form. Today I checked an old plate 
(over a month old) and I see 4 large crystals. *excitement* Three of 
them look tetragonal in shape (like a pyramid) and one of them looks 
hexagonal. I do not know if they are salt or protein. There is calcium 
chloride in the buffer. They feel quite soft to touch. They do not 
cause much birefringence. One of them does not seem to absorb much 
izit. It did go a bit blue but not entirely.


How can I tell if this crystal is protein or not? Do you think its 
worth trying to see how it diffracts?


Also, does Izit affect diffraction/ protein structures at all? Could I 
use a crystal with Izit in a diffraction experiment and ultimately to 
get the structure?


Best
Careina

Re: [ccp4bb] salt or not?

[Ulrike Demmer]

Dear Careina,

altough your crystals does't take up the Izit dye it sounds promising. The 
uptake of Izit depends on the solvent channels of the protein molecule - 
sometimes the dye just can't enter the molecule.
Concerning the Calciumchloride - if the concentration is not too high and 
without other ingredients which could cause less solube salt there is the 
possiblity that you have got protein crystals. I once had a condition whith 34 
% MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting  
crystals
To speak from my experience I think 1 month after setting up the trays the 
drops should not be dried out. If the wells are sealed properly new crytals can 
appear even after 1 year.

You should definately check the diffraction then you will know for sure.

Cheers,

Ulrike

Re: [ccp4bb] salt or not?

[RHYS GRINTER]

What else in in the conditions? Calcium Sulphate/Phosphate is poorly soluble, 
so if there is any sulphate or phosphate in your condition I would be 
suspicious.
The age of the plate is also a bad sign, as evaporation over an extended time 
can lead to salt crystals. Check the well solution for crystals, if there are 
any then it's almost certainly salt.
Softness and lack of birefringence are cautiously good signs, however the only 
way to know for sure is to stick them in an x-ray beam, which is always worth 
while for a crystal.

Good luck

Rhys



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Careina Edgooms 
[careinaedgo...@yahoo.com]
Sent: 15 April 2013 11:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] salt or not?

Dear ccp4

I have been performing trials on a protein DNA complex for a while now and have 
not seen any crystals form. Today I checked an old plate (over a month old) and 
I see 4 large crystals. *excitement* Three of them look tetragonal in shape 
(like a pyramid) and one of them looks hexagonal. I do not know if they are 
salt or protein. There is calcium chloride in the buffer. They feel quite soft 
to touch. They do not cause much birefringence. One of them does not seem to 
absorb much izit. It did go a bit blue but not entirely.

How can I tell if this crystal is protein or not? Do you think its worth trying 
to see how it diffracts?

Also, does Izit affect diffraction/ protein structures at all? Could I use a 
crystal with Izit in a diffraction experiment and ultimately to get the 
structure?

Best
Careina

Re: [ccp4bb] salt or not?

[Hargreaves, David]

Dear Careina,



I would be cautious of using dyes. Much better to 1) try in-situ
diffraction if possible as this is least invasive or 2) pick a sensible
cryo and just freeze the crystal(s). I would try to same something from
the experiment for seed stock & possibly sequencing.



Dave



David Hargreaves

Associate Principal Scientist

_

AstraZeneca

Discovery Sciences, Structure & Biophysics

Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF

Tel +44 (0)01625 518521  Fax +44 (0) 1625 232693

David.Hargreaves @astrazeneca.com 



Please consider the environment before printing this e-mail



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Careina Edgooms
Sent: 15 April 2013 11:18
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] salt or not?



Dear ccp4



I have been performing trials on a protein DNA complex for a while now
and have not seen any crystals form. Today I checked an old plate (over
a month old) and I see 4 large crystals. *excitement* Three of them look
tetragonal in shape (like a pyramid) and one of them looks hexagonal. I
do not know if they are salt or protein. There is calcium chloride in
the buffer. They feel quite soft to touch. They do not cause much
birefringence. One of them does not seem to absorb much izit. It did go
a bit blue but not entirely.



How can I tell if this crystal is protein or not? Do you think its worth
trying to see how it diffracts?



Also, does Izit affect diffraction/ protein structures at all? Could I
use a crystal with Izit in a diffraction experiment and ultimately to
get the structure?



Best

Careina


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