Hi Engin, first off, i would not consider an overall Rmerge of 6-10% lousy data, but quite acceptable for most "real-life", interesting problems (so no lysozyme, thaumatin etc). Our structure of the protein translocation channel SecY is an example of de novo low-res Se phasing (PDB code 1RHZ). Phases were obtained by carefully collected (friedel flipping with 10-20 deg wedges) peak-wavelength SAD datasets combined with cross-crystal averaging to get interpretable maps. We didn't have NCS. The best resolution was about 3.5 A for the selenium datasets. I would say it is possible (but hard) for anything with at least orthorhombic symmetry. I have a Se SAD data set myself that will not give interpretable maps; the symmetry is P1 however and there are multiple lattices, so this seems pretty hopeless...
I would expect there are a bunch of membrane protein structures that fulfill your criteria. Off the top of my head is the small mechano-sensitive channel MscS solved by the Rees group. I think this is 3.9 A data, with a high degree of NCS however. Good luck, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [email protected] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -----Original Message----- From: CCP4 bulletin board on behalf of Engin Ozkan Sent: Sun 5/10/2009 5:01 PM To: [email protected] Subject: [ccp4bb] phasing with se-met at low resolution Hi everyone, I thought I start a new thread while it is unusually quiet on the bb. I am pondering over the practical limitations to MAD and SAD phasing with Se-Met at low resolution. What is the lowest resolution at which people have solved structures "only" using phases from selenium in a "realistic" case? Let me further qualify my question: My *realistic* *low* resolution case is where 1. Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy). 2. Resolution limit is worse than 3.5 Angstroms, where <I>/<sigma> in the last resolution bin is between 1 and 3 (i.e. your crystals are really lousy). 3. Assuming good selenium occupancy (~85%; I work with eukaryotic expression systems, so 100% is not usually achieavable), 4. The number of selenium atoms are enough many that the Crick-Magdoff equation would give you *at least* an average 5% change in intensities (assuming 6 electrons contributed per selenium, based on both absorptive and dispersive differences being at about 6 e- at the absorption edge). 5. and specifically, no other phases and molecular replacement solutions are available. Obviously, I have a case very similar to what's described above, and three years of failure with heavy atom derivatization (I am still trying). I would be happy to hear about Se-Met cases, and data collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing methods used in these cases, or references of them. Again, no other partial phases, and no data cut off at 3.6 A with an I/s of 15 in the last resolution bin. Are there any examples out there? Searching the RCSB and PubMed did not point out to me many successful cases. Thanks, Engin P.S. I would also appreciate the specific query type for searching the PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.). They seem to have everything under the sun searchable, but I cannot find this one.
