Hi Boaz,
In fact, we have a different crystal form. Well, sort of as it is
related to the first form (a=69.1 c=216.7A P6122 vs. 66.6 103.448
P6222). Obviously the second form has only one molecule/ASU. The
interfaces remain the same with the exception that there is no
problematic inconsistency of option 1.
The monomer is not that elongated, but dimer 3 is. We are thinking about
SAXS, as it should distinguish between compact and elongated particle.
But it will take some time before we get beamtime. In the meantime we
can do light scattering and mutations. I am also looking into sequence
conservation.
Thanks,
Karolina
On Thu, 2013-07-25 at 22:53 +0000, Boaz Shaanan wrote:
> Hi Karolina,
>
>
> You have an interesting case in hand but which is not uncommon. From
> your description it sounds as if option 2 is more likely to represent
> the biological dimer but as you noted, it's not certain. Best would be
> to obtain another crystal form (easily said, I know, not easily done).
> Even if this form diffracts poorly compared to the form you have now
> it may give you a clue about which of the 3 dimers is the biological
> dimer. Other options are checking the effect of mutations. Also, since
> you mentioned the elongated form, how sure are that the SEC results
> actually suggest that it's a dimer? If the monomer is elongated and
> has MW of 15kD, running as 24kD is not unheard of. Analyzing SEC
> results can often be tricky when it comes to determining MW and size.
>
>
> My 2p late night thoughts.
>
>
> Boaz
>
>
>
> Boaz Shaanan, Ph.D.
> Dept. of Life Sciences
> Ben-Gurion University of the Negev
> Beer-Sheva 84105
> Israel
>
> E-mail: [log in to unmask]
> Phone: 972-8-647-2220 Skype: boaz.shaanan
> Fax: 972-8-647-2992 or 972-8-646-1710
>
>
>
>
>
>
> ______________________________________________________________________
> From: CCP4 bulletin board [[log in to unmask]] on behalf of
> Karolina Michalska [[log in to unmask]]
> Sent: Friday, July 26, 2013 1:33 AM
> To: [log in to unmask]
> Subject: [ccp4bb] which dimer?
>
>
>
> Hi all,
>
> I'm working with a protein that appears to be a dimer in solution, on
> SEC in runs as 24 kDa, while the actual mass of a dimer is 30. And I
> am trying to figure out which dimer is the biological one (it is a
> regulatory protein but details are uknown). The crystal structure
> gives me a few options (mol A and B in ASU plus P6122 symmetry), but
> none of them is really convincing: for deltaGdiss PISA goes from -0.4
> to 1.5 kcal/mol.
>
> Theoretically, I have two compact dimers (1 and 2) and one elongated
> (3).
>
> In 1, I have two hydrophobic helices interacting and four hydrogen
> bonds, 1550 A2 buried area (out of 14200 total). This interface
> applies only to molecule A and its crystallographic mate, equivalent
> molecules B are too far from each other. Moreover, even in the dimer
> made of mol A there are channel at the interface.
>
> Interface 2 is purely hydrophobic, but at least it's consistent, i.e
> there are comparable interactions for A-A and B-B pairs, 1850 A2
> buried area
>
> Interface 3 involves non-crystallographic copies, buried area is 1040
> A2. The interacting elements are proline-rich, and there are four
> main-chain - main chain hydrogen bonds plus two main-chain - side
> chain ones. Formally, these fragments are not classified as
> beta-strands, but the association does look like an intermolecular
> beta-sheet. This dimer is not consistent with the SEC data though. I'm
> assuming that with an elongated shape it would run as a bigger
> particle than it actually is, not as a smaller one.
>
> So I think I can discard first option but I am still debating on 2 and
> 3. I'll appreciate your comments on this.
>
> Karolina
>
>
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