Dear Appu,
A lot of things may happen when you try to soak: Crystals may dissolve,
crack, the substrate may not bind or get turned over, or the experiment
may just work. The first thing to try is to just do the experiment. You
may want to increase the precipitant concentration somewhat, say from 20
to 25% peg to reduce the risk that the crystal will crack or dissolve.
To prevent turnover, if your enzyme needs a cosubstrate, say NADH, you
could leave that out, or you could try to soak in the reaction product
or a substrate analog which cannot be converted by the enzyme.
However, if you suspect there might be a conformational change in your
protein, this can be prevented by the crystal packing and you may end up
with the substrate bound to the open form of the enzyme. In general, the
gold standard is to cocrystallize your enzyme with the
substrate/product/analog under conditions where there is no turnover and
that is something you definitively should try.
To finally come to your question: you may indeed end up with a mixture
of apo and bound enzymes. For the processing, that makes no difference.
The crystal may be cracked and diffract badly, or it may just be fine.
If there is no conformational change, your substrate will have an
occupancy lower than 1 and weak electron density. During refinement you
have to correct for it but otherwise everything should be fine. If there
is a conformational change, your electron density maps may look quite
chaotic, being a mixture of apo and substrate bound conformations and
you may have to fit alternative postions for a significant numbers of
residues. Again, cocrystallization is the way to go.
There are two tricks to get as much of your active sites occupied as
possible:
1) use the highest possible substrate concentration, which may be as
high as 100 mM (check the pH of the solution before you add it to the
crystal!)
2) Another trick, especially if you get some slow turnover, is to add
fresh substrate in a high concentration shortly before you freeze, and
add the substrate to your cryoprotectant solution, otherwise your
substrate may be gone in seconds!
Good luck!
Herman
________________________________
From: CCP4 bulletin board [mailto:[email protected]] On
Behalf Of Appu kumar
Sent: Wednesday, December 12, 2012 6:45 PM
To: [email protected]
Subject: [ccp4bb] pathological case with soaking
Respected members,,
I have one question
bearing in my mind regarding soaking. Binding of the substrate to enzyme
leads to active site closure. So if we are soaking the enzyme crystal
with the substrate, and collecting the data and solving the structure,
then how would one distinguish the existence of apo and substrate bound
form of enzyme in same crystal. I meant to say, soaking may cause the
crystal to have quasi-apo-enzyme and quasi-substrate-enzyme complex
existing together. Is there any way to take care of such pathological
condition during data processing, or structure solving? . I would highly
appreciate your help and looking forward to get valuable advices. Please
graduate me in this kind of weired situation.
Thank you
Appu
