Dear Prerana,
Since in your post you do not mention pH, perhaps you should
consider buffers and pH in your protocol to improve the diffraction of
your crystals.
I suggest that you do so in 3 steps:
Step 1:
While retesting the same crystals you have tested but
with mixed cryoprotectants at different pH using linear buffers that will
allow you to change pH without altering
the buffer components: (Different mixtures and different pH give different
results)
Newmann J. (2004) Novel buffer system for macromolecular crystallization,
Acta Cryst. D 60, 610-612.
145. Vera L., Stura E.A. (2014) Strategies for protein
cryocrystallography. Crystal Growth & Design, 14: 427-435.
148. Ciccone L., Tepshi L., Nencetti, S. & Stura E.A. (2015) Transthyretin
complexes with curcumin and bromo-estradiol: Evaluation of solubilizing
multicomponent mixtures New Biotech. 32:54-64.
149 Ciccone L., Vera L., Tepshi L., Rosalia L., Rossello A. & Stura E.A.
(2015) Multicomponent mixtures for cryoprotection and ligand
solubilization. Biotechnology Reports 7:120—127.
There are kits for both the linear buffers and the mixed cryoprotectants.
Step 2:
The pH should be varied in coarse steps. Make a note at which pH you get
the best diffraction.
If you do not get spontaneous nucleation use seeding.
19. Stura E.A., Wilson I.A. (1991) Applications of the streak seeding
technique in protein crystallization. J. Cryst. Growth 110:270-282.
75. Stura E.A. (1999) Strategy 3: Reverse Screening. In "Crystallization
of Proteins: Techniques, Strategies and Tips. A laboratory manual"
(Bergfors, T., ed) International University Line. pp. 113-124.
Step 3:
The pH should be varied in fine steps with different buffers.
Enrico.
Ask for a PDF if you do not have access to any of the above papers.
On Wed, 20 Jan 2016 06:03:20 +0100, Prerana G. <[log in to unmask]> wrote:
> Dear all,
>
> I have a question on how to improve the resolution of protein crystals. I
> am working on a protein which crystallizes in the following conditions
>
> 1) 0.1M Magnesium nitrate hexahydrate & 10% PEG 3350
>
> 2) 0.2M Calcium chloride dihydrate & 6-14% PEG 3350
>
> 3) 0.2M Calcium acetate hydrate & 6-14% PEG 3350
>
> The crystals diffracted to a very low resolution (~ 4 Angst, kindly see
> the
> attached image) at home source. To prevent ice formation, different
> cryoprotectants such as glycerol, 2-methyl-2,4-pentanediol, PEG 400, PEG
> 3350, Hexanediol, were used.
>
> Any suggestion to improve the x-ray diffraction resolution will greatly
> help me.
>
>
>
> Thanking you.
>
> Prerana
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
e-mail: [log in to unmask] Fax: 33 (0)1 69 08 90 71
Proxima-2A, Soleil Synchrotron. Tel: 33 (0)1 69 35 8180 Beamline
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