Dear Nishant,
To me, the diffraction looks like powder diffraction from a salt. In
your case, I would look at the protein buffer to see if there are
components present (phosphate, detergent etc.) which might be prone to
form crystals on their own and see if you can find a minimal buffer
where the protein is still happy. With this buffer, I would do another
full screening to see if you get hits with more protein-like
diffraction.
Best,
Herman
________________________________
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Niks
Sent: Tuesday, July 24, 2012 4:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Improvement in crystal quality
Dear All,
I am trying to crystallize a recombinant dehydrogenase protein.
Got five hits in PEG ION Screen from Hamptons (20% PEG 3350 with 0.2M
Sodium Acetate, 0.2M Potassium acetate, 0.2M ammonium acetate, 0.2M
sodium formate and 0.2M Potassium Chloride) after two days.
Crystals looks like needles most of time, sometime broader
needles (Pictures attached). UV crystal scanner says those are protein
crystals, but when we tried to pick up one and shoot at room
temperature, diffraction patterns looks like similar like of powder
diffraction (picture attached).
I have tried 50 of the 96 additives(whichever I can arrange of)
mentioned in the additive screen from Hamptons . I have tried detergent
screen from Hamptons (this time original screen solutions). I have tried
incubating the plate at 28degrees as well as 10degrees, Though waiting
for 10degree results but one drop showed needles again after normal two
days of growth period.
I tried to slow down the supersaturation by adding 100ul of 1:1
ratio of silicon oil and paraffin oil over the 1ml of well solution.
This time no crystals but some precipitation.
If anyone spare any word of wisdom to improve these crystal
quality, I will be very grateful.
If seeding is the only obvious thing to try, any reference for
the seeding procedure will be highly appreciated.
Thanks very much
Nishant Varshney
PhD student,
National Chemical Laboratory,Pune,India
--
"The most difficult phase of life is not when No one
understands you;It is when you don't understand yourself"
--
"The most difficult phase of life is not when No one
understands you;It is when you don't understand yourself"
