First, I wouldn't worry about the extent of SeMet incorporation. If you have followed an established protocol (either using an auxotroph or regular strain) the incorporation should be fine.
For your SeMet data, what is the redundancy and resolution? In my experience it is often very hard to find Se sites and solve the structure for low resolution data (lower than 3 A). Most often the data is just not good enough to pick out the weak signal, and, like in your case, you get "useful" anomalous signal to 5-6 A resolution. However, you should also try different programs like SOLVE and AUTOSOL within phenix. Also, given that you have a high symmetry space group, it may be possible to remove some data and still have sufficient redundancy; check carefully if you have radiation damage and cut off data accordingly. For phase extension you first need to have decent phases....... Good luck, bert ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of vijay srivastava [vijaytec...@yahoo.co.in] Sent: Tuesday, June 03, 2014 9:37 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] experimental phasing at low resolution Dear All, Does anyone know of a facility that provides amino acid analysis for determination of SeMet incorporation in a recombinant protein? We're looking for some advice about how to proceed with a structure we're working on. Our protein is 350 amino acids (10 methionine are present) and naturally binds magnesium. We have a SeMet data set at peak that goes down to 5.4 angstroms. We tried to find the heavy atom position with Shelxcde program. The log file is given as below. Resl. Inf - 8.0 - 6.0 - 5.6 - 5.4 - 5.2 - 5.0 - 4.8 - 4.6 - 4.4 - 4.2 - 3.98 N(data) 529 643 241 29 0 0 0 0 0 0 0 <I/sig> 63.5 19.5 10.6 10.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 %Complete 94.1 98.9 99.2 18.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 <d"/sig> 3.06 1.24 1.07 1.21 I am also attaching the .lst and .res file and from that we were considering that only one selenomethionine position.We have one monomer per AU (-unfortunately, MR is not working for this project). The space group is P3 . We also have a native set down to 3.4 angstroms. While we understand that we may need more phasing information we're wondering if anyone might have some other suggestions or insights about how we can move forward given the data that we currently have. Can we extend the phases with our native data set. Thanks in advance for any advice. regards Vijay
