Dear Bishwa,
The first thing I would try is to add a ligand (natural, inhibitor etc.). This may solve your problem. Also, why do you have 400 mM KCl in the storage buffer? Is it because the protein would otherwise aggregate/precipitate? Or is it because it came off the last purification column like that? I would try to reduce the amount of salt to at least 100-150 mM.
Also, if the crystals can be mounted, I would test a large number at a synchrotron. Often there many, many crystals with bad diffraction and suddenly one which diffracts to much higher resolution. What kind of resolution do you get from your crystals?
Best,
Herman
-----Ursprüngliche Nachricht-----
Von: CCP4 bulletin board [mailto:[log in to unmask]] Im Auftrag von Bishwa Subedi
Gesendet: Donnerstag, 30. Juni 2016 02:11
An: [log in to unmask]
Betreff: [ccp4bb] Improving the crystals
Hi All,
I have been obtaining a multiple thin plates crystals attached to each other on the surface of the sitting drop. I have tried additive and silver bullet screen from Hampton to see if they improve, but all my hits in the new screen are again almost the same. pH optimization around the initial hit (5.5 and 6.5) also gives similar crystals within the range. Also the rMMS method gave crystals of similar morphology on several wells. I would appreciate your tips on improving this crystal.
P.S. the protein is ~50 kDa, pI 9.6, and has 20 mM MOPS and 400 mM KCL in the storage buffer (pH 7.0). Protein concentration used in this experiment is 9.0 mg/mL
Thank you,
Bishwa
|