Re: [ccp4bb] Expert opinion for optimizing ethylene glycol crystallization condition

2013-01-04 Thread Sankaranarayanan Srinivasan 
Dear all,

Thank you very much for all your helpful comments. I will try them and post
on the BB my results.

Best regards

Shankar

On Wed, Jan 2, 2013 at 11:59 AM, Sankaranarayanan Srinivasan 
texs...@gmail.com wrote:

 Dear all,

 A very happy new year to all.

 I would appreciate some expert advice on optimizing a crystallization
 condition in which the initial hits were obtained with ethylene glycol as
 the main precipitant. Here is the summary of things tried.

 We have a protein, size (31Kda) and the starting protein buffer is 0.1M
 Tris pH7.5, 0.1M NaCl, 10% glycerol.
 The initial crystal hit was obtained from the emerald cryo kit condition
 that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals
 were tiny (10-20um). A crystallization matrix to obtain better crystals
 by varying the imidazole pH and ethylene glycol concentrations was tried
 from which the best condition obtained was 0.1M imidazole pH 6.5 , 30%
 (v/v) ethylene glycol. The crystals were slightly bigger 50um.
 On trying the additive screen, bigger crystals (200um) were obtained, but
 putting them under the x-ray beam with direct freezing did not yield any
 diffraction spots.
 Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture
 also yielded similar results.
 Low resolution spots near the beam stop were also not seen. Similarly
 spots indicative of salt was also not seen. It just had hazy ice rings kind
 of stuff. (The beam was definitely on the crystal)
 To check if what we have was salt, a control condition with no protein was
 tried. Also the crystals were run on a gel after thorough washing. Both
 these tests, show that they are definitely protein crystals and not salt.
 Seeding also did not yield any improved crystals.
 I was suggested using di-ethylene glycol, propane diol as alternatives.
 I would greatly appreciate if you can give your opinion on using other
 di-alcohols as precipitants or other ways to improve these crystals.
 I tried searching the PDB to see if someone had actually used ethylene
 glycol as a precipitant, most of them were used as a cryo condition than
 actually as a precipitant.

 Thank you very much in advance.

 Regards
 Shankar Srinivasan

[ccp4bb] Expert opinion for optimizing ethylene glycol crystallization condition

2013-01-02 Thread Sankaranarayanan Srinivasan 
Dear all,

A very happy new year to all.

I would appreciate some expert advice on optimizing a crystallization
condition in which the initial hits were obtained with ethylene glycol as
the main precipitant. Here is the summary of things tried.

We have a protein, size (31Kda) and the starting protein buffer is 0.1M
Tris pH7.5, 0.1M NaCl, 10% glycerol.
The initial crystal hit was obtained from the emerald cryo kit condition
that has 0.1M imidazole pH 8.0 , 50% (v/v) ethylene glycol. The crystals
were tiny (10-20um). A crystallization matrix to obtain better crystals by
varying the imidazole pH and ethylene glycol concentrations was tried from
which the best condition obtained was 0.1M imidazole pH 6.5 , 30% (v/v)
ethylene glycol. The crystals were slightly bigger 50um.
On trying the additive screen, bigger crystals (200um) were obtained, but
putting them under the x-ray beam with direct freezing did not yield any
diffraction spots.
Trying other cryo-conditions like glycerol and 50-50 paratone/oil mixture
also yielded similar results.
Low resolution spots near the beam stop were also not seen. Similarly spots
indicative of salt was also not seen. It just had hazy ice rings kind of
stuff. (The beam was definitely on the crystal)
To check if what we have was salt, a control condition with no protein was
tried. Also the crystals were run on a gel after thorough washing. Both
these tests, show that they are definitely protein crystals and not salt.
Seeding also did not yield any improved crystals.
I was suggested using di-ethylene glycol, propane diol as alternatives.
I would greatly appreciate if you can give your opinion on using other
di-alcohols as precipitants or other ways to improve these crystals.
I tried searching the PDB to see if someone had actually used ethylene
glycol as a precipitant, most of them were used as a cryo condition than
actually as a precipitant.

Thank you very much in advance.

Regards
Shankar Srinivasan

Re: [ccp4bb] Protein melting temperatures

2011-09-28 Thread sankaranarayanan srinivasan 
This paper on thermofluor is a good reference and if you have access to a
real time PCR machine, different buffer systems, like the PACT screen can be
evaluated within an hour to find out the buffer in which your protein is
most stable.
It gives the Tm of your protein and if you have a high fluorescence to start
with, it means your protein is unfolded to start with.

Curr Protoc Mol Biol. http://www.ncbi.nlm.nih.gov/pubmed/21472694# 2011
Apr;Chapter 10:Unit10.28.
The combined use of the Thermofluor assay and ThermoQ analytical software
for the determination of protein stability and buffer optimization as an aid
in protein crystallization.
Phillips 
Khttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Phillips%20K%22%5BAuthor%5D
, de la Peña 
AHhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22de%20la%20Pe%C3%B1a%20AH%22%5BAuthor%5D
.

Best regards

Shankar

On Wed, Sep 28, 2011 at 11:03 AM, Linda Schuldt lschu...@mb.au.dk wrote:

 Dear Raji,

 what exactly do you mean when you say the melting temperature is 45deg.
 Did you only test one buffer, or did you test many buffers and 45deg is
 the most stable one? If you have only tested one buffer you should run a
 screen testing different buffer systems (pH) and e.g. NaCl concentration
 and glycerol concentrations (or ligands, if your proteins binds any). Then
 you identify the buffer which is stabilizing your protein the most. I have
 seen big impacts on protein stability and crystallization when optimizing
 my buffers like this.

 I think you should not only consider the melting temperature alone, but
 also how the curve looks like. Do you get a high initial flourescence
 (which often indicates partially unfolded protein or hydrophobic patches)
 or do you have very low initial flourescence (which is a good sign for
 compact protein). Another thing to look at is if your transition is sharp
 (the steeper the better). Taking all this together you can judge if your
 protein is happy or not.

 Hope this helps you!

 Linda

 Patrick Shaw Stewart wrote:
  I actually think you *can *make comparisons between different proteins.
  We
  heard a very nice talk by Jose Marquez about exactly this at the RAMC
  meeting recently.
 
  Basically, 45C seemed to be the dividing line.  If your protein melts
  below
  this it's a bad sign for crystallization and may point to setting up your
  crystallization experiments at lower temperatures.
 
  Patrick
 
 
 
  On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis
  a.perra...@nki.nlwrote:
 
  **
 
  Hello -
 
  The excellent paper of McCrary, uses differential scanning
  calorimetry, which will give an absolute measure of thermostability.
 
  Using Thermofluor I would be afraid you can only assess the relative
  thermostability of one protein in different conditions.
  As your fluorescence reporter would interact differently with exposed
  hydro[hobic patches in different proteins, I would be a bit more careful
  in comparing the Thermofluor results between different proteins ... I
  am not aware of anyone correlating differential scanning calorimetrywith
  Thermofluor data, but I must admit I have not looked up that
  literature recently.
 
  A.
 
 
  On 23 Sep 2010, at 18:40, Philippe DUMAS wrote:
 
   Le 23/09/2010 17:28, Raji Edayathumangalam a écrit :
  
   Raji
   I suggest having a look to this paper:
   McCrary et al. J. Mol. Biol. 264(1996) 784
   where you will find an interesting study on protein stability and an
   interesting comparison with other proteins.
   Philippe Dumas
  
   Hi Folks,
  
   Sorry for the pre-xtallo question; pre-xtallo right now, but hoping
   to
   take my protein the xtallo way one of these days!
  
   I am currently performing Thermofluor assays with my protein and the
   results show that the Tm is ~45C.  I am looking for some examples of
   proteins and their melting temperatures so that I can gauge where my
   protein falls in the spectrum of unstable-to-stably folded. For
   example, the melting temperature of some forms of lysozyme is 73.8C
   (very stable, I suppose).
  
   Just need a sense for whether my protein is considered unstable or
   somewhat stable. Please could you share some examples.
  
   Many thanks.
   Raji
  
   ---
   Raji Edayathumangalam
   Joint Research Fellow
   Harvard Medical School/
   Brigham and Women's Hospital
   Brandeis University
  
  
   McCrary-JMB264(1996)784.pdfp_dumas.vcf
 
 
 
 
  --
   patr...@douglas.co.ukDouglas Instruments Ltd.
   Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
   Directors: Peter Baldock, Patrick Shaw Stewart
 
   http://www.douglas.co.uk
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 ***
 Dr. Linda Schuldt
 Department of Molecular Biology
 University of Aarhus
 Science Park
 Gustav Wieds Vej 10c
 DK-8000 Århus C
 Denmark