Rhys, Having worked on a number of these structures using both MR and SAD/MAD, my advice is to continue to make experimental phasing a priority since building into a poor density map at this resolution can take months to years to finish while building into a nice experimental map can take a few hours to days, depending on how good your phases are. With that being said, I fully support pursuing the MR route, just keep in mind though it won't easy at this resolution, even if you had awesome data. And yes, your results seem to indicate that you have a nice solution, but i wonder how much of the overall structure does your model cover? You mention this is a 22-stranded beta-barrel....membrane protein? A tonB-dept transporter maybe....and if so, do you get a solution with the plug domain or are you just playing with the barrel domain currently? A good check to see if you have a real solution if you are only using the barrel domain is to check to see if you have any difference density for the plug domain.
So, my 2 cents, keep chugging along with the MR, but I would put more energy into the experimental phasing, esp if you are able to express it ok and can crystallize using the same conditions as native. Also, both MR and experimental phasing would benefit greatly if you were able to increase your resolution closer to 3 angstroms (ugh....i know...easier said than done!). Again, if this is indeed a membrane protein, that could just mean that you end up screening a few hundred crystals to find that one crystal with that one sweet spot that gives you that one really good dataset. Good luck with your project, sounds like you are almost there! Cheers, Nick +++++++++++++++++++ Nicholas Noinaj Laboratory of Molecular Biology NIDDK, NIH 50 South Drive, Room 4505 Bethesda, MD 20892-8030 Phone (+1) 301-594-9230 Web: http://www-mslmb.niddk.nih.gov/buchanan/index.html ++++++++++++++++++++ ________________________________________ From: RHYS GRINTER [r.grinte...@research.gla.ac.uk] Sent: Monday, September 23, 2013 5:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] tricky mr problem Hi all, I have been attempting to find a MR solution for a low resolution data set (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel I'm working on. I've created a trimmed poly-alanine from a structure of 17% identity, that gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 900). I'm guessing this in a genuine solution, but the map is too poor to build into. Does anyone have any advice as to proceed from here? It may be just a case of needing better resolution data to work with, but would this indicate that Selenomet derivative crystals won't be needed for this structure? Cheers, Rhys
