mixture with unknown component

Linear (OLIGOMER), and non-linear (MIXTURE) analysis, singular value decomposition (SVDPLOT), addition of missing fragments (BUNCH, CORAL), analysis of flexible systems (EOM/RANCH & GAJOE), flexible refinement of high-resolution models (SREFLEX)
Post Reply
Message
Author
gia75
Member
Posts: 13
Joined: 2014.05.20 18:09

mixture with unknown component

#1 Post by gia75 » 2014.06.30 15:21

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

Alex
Active member
Posts: 692
Joined: 2007.08.09 21:10
Location: Planet Earth

Re: mixture with unknown component

#2 Post by Alex » 2014.07.01 10:47

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

gia75
Member
Posts: 13
Joined: 2014.05.20 18:09

Re: mixture with unknown component

#3 Post by gia75 » 2014.07.01 12:05

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
Attachments
gnom.png
gnom.png (17.73 KiB) Viewed 3812 times
curve.png
curve.png (21.01 KiB) Viewed 3812 times

Alex
Active member
Posts: 692
Joined: 2007.08.09 21:10
Location: Planet Earth

Re: mixture with unknown component

#4 Post by Alex » 2014.07.01 12:09

what does the MW estimation say?

gia75
Member
Posts: 13
Joined: 2014.05.20 18:09

Re: mixture with unknown component

#5 Post by gia75 » 2014.07.01 12:39

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)

Alex
Active member
Posts: 692
Joined: 2007.08.09 21:10
Location: Planet Earth

Re: mixture with unknown component

#6 Post by Alex » 2014.07.01 12:43

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?

gia75
Member
Posts: 13
Joined: 2014.05.20 18:09

Re: mixture with unknown component

#7 Post by gia75 » 2014.07.02 15:21

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

Alex
Active member
Posts: 692
Joined: 2007.08.09 21:10
Location: Planet Earth

Re: mixture with unknown component

#8 Post by Alex » 2014.07.02 15:42

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

Post Reply