Re: [ccp4bb] which dimer?

[Boaz Shaanan]

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: bshaa...@bgu.ac.il Phone: 972-8-647-2220  Skype: boaz.shaanan                  Fax:   972-8-647-2992 or 972-8-646-1710                          From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Karolina Michalska [dzi...@amu.edu.pl] Sent: Friday, July 26, 2013 1:33 AM To: CCP4BB@JISCMAIL.AC.UK 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