Dear EnginI would also like to comment. I our recent structure determination of the sodium pump (3.5 A) (see morth JP et al 2007) we did not have experimental phasing to more than 6 A for the Ta6Br12 clusters and 7 A for the Pt sites. Both with extensive multicrystal averaging and phase combination it was possible to trace the structure. The data was what you could call lousy, but in the end I was able to identify the 3 Rubidium ions present in the data set based on the their anomalous scattering power. Not detectable in the reflection statistics of the native data set (see Schack VR et al 2008). So even though your selenium sites will not help the phasing initially, they will still guide the model building extensively (see Hunte C et al 2005 Nature, 3.5 A res structure with Se) with your improved model phases the selenium site positions will improve and at a later stage they will be valuable, when combined as additional phase information. The initial maps always look really bad, your 3 years of heavy atom derivatisation might not have been in vain, you can still use the initial Se phases to try to locate more heavy atoms sites (Fredslund F et al 2006 jmb)
good luck Preben
On 11/05/2009, at 05.24, Engin Ozkan wrote:
Wow, I got quite a number of responses. Thanks everyone. Let's elaborate.Petr, I don't know my anomalous signal, because I haven't yet done the experiment. If I had, I would have definitely talked about chi2 values for I+/I- merged and unmerged, Anomalous Patterson maps, or other measures of anomalous signal (Dauter, Acta Cryst D, 2006 is a great paper to read on that, I suggest every grad student to present it in their Crystallography Journal Club). I was merely pondering today, but I plan to do the experiment very soon (crystals are waiting for synchrotron time).About your example, 2.9 A diffracting crystals (with 4 A anomalous signal) are in the doable range, as you suggested. The question is what would happen if your crystals diffract to 4 A, and anomalous signal dies at 6 A. The interesting bit of course is 1 Met per 200 residue, which should put to death the "1 in 50" or "1 in 100" Methionine myths: it depends on the quality of your data.Engin On 5/10/09 2:50 PM, Leiman Petr wrote:Dear Engin Ozkan,You have told us how bad your crystals are, but you did not mention how good your anomalous signal is: 1. To what resolution does your anomalous signal extend and what statistic is used for this estimate? 2. Do your dispersive and Bijvoet Pattersons look similar and what is the measure of similarity?This structure http://www.pdb.org/pdb/explore/explore.do?structureId=1K28which contains ~1100 residues in the asymmetric unit (and ~3500 in the entire complex), was solved using a chimerical SeMet derivative, in which one protein was SeMet labeled (17 Se per a.u.) and the other was native. The Semet dataset had a detectable anomalous signal to 4 A resolution (at most). The diffraction extended to 2.9A resolution.Sincerely, Petr ----------------------------------------------------------- Petr Leiman Institut de physique des systèmes biologiques École Polytechnique Fédérale de Lausanne (EPFL) Cubotron/BSP-415 CH-1015 Lausanne Switzerland-----Original Message-----From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf OfEngin Ozkan Sent: Sunday, May 10, 2009 11:01 PM To: CCP4BB@JISCMAIL.AC.UK 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 peoplehave 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> inthe 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 absorptiveand 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 otherpartial phases, and no data cut off at 3.6 A with an I/s of 15 in thelast resolution bin. Are there any examples out there? Searching the RCSB and PubMed did not point out to me many successful cases. Thanks, EnginP.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 seemto have everything under the sun searchable, but I cannot find this one.
Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/da/j...@mb
