Page 1 of 1

mixture with unknown component

Posted: 2014.06.30 15:21
by gia75
Hi All,

I have a 40kD protein that make nice hexamers in solution (we have the crystal structure + secmalls data).
However, the SAXS data show the presence of additional component, which (we believe) could be a dodecamer.
We don't understand yet how the dodecamer could be formed as we only see hexamers in the crystal structure and the symmetry does not help.
Now, how can I model such a system ?
Can I calculate separate envelops for the 2 components ?
Thanks for your suggestions,
GIA

Re: mixture with unknown component

Posted: 2014.07.01 10:47
by Alex
Now, how can I model such a system ?
if the structure of a hexamer is complete, I think you can try using SASREF MX.
Can I calculate separate envelops for the 2 components ?
You may try GASBOR MX, if the protein is not too large (40kDa should actually be ok).

i would also consider separating it better, say using inline HPLC-SAXS.

Best, Alex

Re: mixture with unknown component

Posted: 2014.07.01 12:05
by gia75
hi
thank for the reply
we are already using online hplc and we get only 1 pic
the problem is that the Rg and Dmax are much bigger than what we expect
for our hexamer (max dimension 110A) ... i post data and gnom
Best
GIAX

Re: mixture with unknown component

Posted: 2014.07.01 12:09
by Alex
what does the MW estimation say?

Re: mixture with unknown component

Posted: 2014.07.01 12:39
by gia75
if I compare it to another protein (170kDa, monomer) that
I have measured the same day, I get 440kDa for this one.
twice the MW of the hexamer (220kDa)

Re: mixture with unknown component

Posted: 2014.07.01 12:43
by Alex
if you have a crystal structure, try to fit the data using just hexamer (CRYSOL) or a dimer of hexamers (program SASREF).
What fits better?

Re: mixture with unknown component

Posted: 2014.07.02 15:21
by gia75
sorry for bothering with lots of questions.
i have problem to figure out the symmetries in the sasref server.
if my hexamer is symmetry P32, what should be the dodecamer ? (as i can't tell -a priori- if it is a real dimer of hexamers)
or should I take the problem the other way around: dodecamer that dissociates into hexamers ?
thank you

Re: mixture with unknown component

Posted: 2014.07.02 15:42
by Alex
sasref server.
I am not sure if SASREF MX is available as a server.
dodecamer that dissociates into hexamers ?
given you have a crystal structure of hexamer - i think easiest is to consider
it as a monomer and assume it associates into dimers (P2; dodecamers).

Best, Alex