Re: [ccp4bb] Intergrown crystals and excess of nucleation

[Diana Tomchick]

In addition to all the excellent suggestions to perform seeding, you could also 
try sitting drop vapor diffusion, and also different temperatures. Sometimes 
moving from 20 to 4 degrees does the trick.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Sep 9, 2019, at 10:22 AM, Nikolas 
mailto:nikolas.ca...@gmail.com>> wrote:

Dear crystalgrowers,

I am currently working with a protein that appeared to be friendly but turned 
out it was not the case.
I found myself to face -in the scale up- the opposite of the usual problem of 
nucleation (I really love how this topic finds new ways to make fun of me). In 
24-well plates, hanging-drop, for the same condition but in different drops I 
found few big but intergrown crystals and/or a full with microcrystals. 
Sometimes also in the same well, when having more drops.
I already decreased the concentration to less than 4mg/mL, made small 
adjustments in the optimizations - both with apo and ligand samples, used Al's 
oil.

I have read about the "containerless crystallization" but since I cannot obtain 
the sample myself I would like to know if there are any experiences and/or if 
there are suggestions for solving this problem.

Many thanks!

Best regards,
Nikolas



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Re: [ccp4bb] Intergrown crystals and excess of nucleation

[Patrick Shaw Stewart]

Hi Nikolaus

I completely agree with Claude's comments.  Microseeding is the first thing
to try.  You would like to find conditions where you *don't *get crystals
without seeding, but you *do *get crystals with seeding.  Then, just by
diluting the seed stock, you can control nucleation.  Look at D'Arcy and
Obmolova's papers, links below (second time!).

I also agree that crystallization in agarose can work well - if seeding
doesn't solve your problem.

The only person that I know who tried containerless crystallization ended
up with more crystals rather than fewer.

Actually your case is not very unusual - when you scale up you tend to
get *more
*crystals.  The reason is that the surface-area-to-volume ratio is greater
for smaller drops.  This means that you lose a greater proportion of the
protein on the plastic or glass surface of your plate, and also on the
air-drop interface.  Therefore you should dilute the protein and/or the
precipitant when you scale up.

Good luck, Patrick

http://scripts.iucr.org/cgi-bin/paper?S2053230X14015507
https://scripts.iucr.org/cgi-bin/paper?nj5193



On Mon, Sep 9, 2019 at 4:45 PM Claude Sauter 
wrote:

> Le 09/09/2019 à 17:22, Nikolas a écrit :
>
> Dear crystalgrowers,
>
> I am currently working with a protein that appeared to be friendly but
> turned out it was not the case.
> I found myself to face -in the scale up- the opposite of the usual problem
> of nucleation (I really love how this topic finds new ways to make fun of
> me). In 24-well plates, hanging-drop, for the same condition but in
> different drops I found few big but intergrown crystals and/or a full with
> microcrystals. Sometimes also in the same well, when having more drops.
> I already decreased the concentration to less than 4mg/mL, made small
> adjustments in the optimizations - both with apo and ligand samples, used
> Al's oil.
>
> I have read about the "containerless crystallization" but since I cannot
> obtain the sample myself I would like to know if there are any experiences
> and/or if there are suggestions for solving this problem.
>
> Many thanks!
>
> Best regards,
> Nikolas
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> Dear Nikolas,
>
> since you have some crystal stock, I would definitely try seeding to
> better control nucleation events in your drops. Then, instead of using the
> containerless approach which requires two types of oils to prepare floating
> drops, I suggest the crystallization in agarose gel. Easy to perform, it
> favors the 3D growth of well separated crystals in ideal convection-less
> conditions. In addition, the gel provides a physical protection of your
> crystals during handling, mounting and cryocooling. For more details, see 
> "Crystal
> growth of proteins, nucleic acids, and viruses in gels. Lorber et al. Prog.
> Biophys. Mol. Biol. (2009), 101: 13-25."
>
> Happy crystallization!
>
> Claude
>
> --
> Dr Claude Sauter
> Institut de Biologie Moléculaire et Cellulaire (IBMC-ARN-CNRS)
> Biologie des ARNt et pathogénicité  tel +33 (0)388 417 102
> 2 allée Conrad Roentgen fax +33 (0)388 602 218
> F-67084 Strasbourg - France  http://cj.sauter.free.fr/xtal
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
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Re: [ccp4bb] Intergrown crystals and excess of nucleation

[Claude Sauter]

  
  
Le 09/09/2019 à 17:22, Nikolas a
  écrit :


  
  
Dear
crystalgrowers,
  
  
  I am
currently working with a protein that appeared to be
friendly but turned out it was not the case.
  I found
myself to face -in the scale up- the opposite of the usual
problem of nucleation (I really love how this topic finds
new ways to make fun of me). In 24-well plates,
hanging-drop, for the same condition but in different drops
I found few big but intergrown crystals and/or a full with
microcrystals. Sometimes also in the same well, when having
more drops.
  I already
decreased the concentration to less than 4mg/mL, made small
adjustments in the optimizations - both with apo and ligand
samples, used Al's oil. 
  
  
  I have
read about the "containerless crystallization" but since I
cannot obtain the sample myself I would like to know if
there are any experiences and/or if there are suggestions
for solving this problem.
  
  
  Many
thanks!
  
  
  Best
regards,
  Nikolas

  
  
  
  To unsubscribe from the CCP4BB list, click the
following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
  

Dear Nikolas,
since you have some crystal stock, I would definitely try seeding
  to better control nucleation events in your drops. Then, instead
  of using the containerless approach which requires two types of
  oils to prepare floating drops, I suggest the crystallization in
  agarose gel. Easy to perform, it favors the 3D growth of well
  separated crystals in ideal convection-less conditions. In
  addition, the gel provides a physical protection of your crystals
  during handling, mounting and cryocooling. For more details, see "Crystal growth of proteins, nucleic acids, and
viruses in gels. Lorber et al. 
  Prog. Biophys. Mol. Biol.  (2009),
  101: 13-25."
Happy crystallization!
Claude

-- 
Dr Claude Sauter
Institut de Biologie Moléculaire et Cellulaire (IBMC-ARN-CNRS)
Biologie des ARNt et pathogénicité  tel +33 (0)388 417 102
2 allée Conrad Roentgen fax +33 (0)388 602 218
F-67084 Strasbourg - France  http://cj.sauter.free.fr/xtal

  



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[ccp4bb] RE [ccp4bb] Intergrown crystals and excess of nucleation

[Andrew Lovering]

Hi Nikolas

The first thing I would try is to solubilise the protein a bit more, maybe by 
adding a few % (1-5%) glycerol or ethylene glycol to the growth conditions when 
you set up the drops.

The next thing would be to experiment with ratio (protein:precipitant) and drop 
size.

Lastly, trial addition of detergents to conditions.

Best!
Andy



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[ccp4bb] Intergrown crystals and excess of nucleation

[Nikolas]

Dear crystalgrowers,

I am currently working with a protein that appeared to be friendly but
turned out it was not the case.
I found myself to face -in the scale up- the opposite of the usual problem
of nucleation (I really love how this topic finds new ways to make fun of
me). In 24-well plates, hanging-drop, for the same condition but in
different drops I found few big but intergrown crystals and/or a full with
microcrystals. Sometimes also in the same well, when having more drops.
I already decreased the concentration to less than 4mg/mL, made small
adjustments in the optimizations - both with apo and ligand samples, used
Al's oil.

I have read about the "containerless crystallization" but since I cannot
obtain the sample myself I would like to know if there are any experiences
and/or if there are suggestions for solving this problem.

Many thanks!

Best regards,
Nikolas



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