I also forgot to mention that I also have a complete dataset at a remote wavelength (shorter) so I can also take advantage of f' differences. I did not collect three wavelengths though. I guess what I really would like to do is to derive experimental phases from the two wavelengths and the arrangement of Yb and S atoms in the structure. I would like to just provide the known coordinates for each and then derive phases that I can then use as restraints in the refinement of the structure at the first wavelength (longer wavelength). I know how to do this the old way but how do I do it in Phenix. ______________________________________________________________________________________ Joseph P. Noel, Ph.D. Arthur and Julie Woodrow Chair Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: [email protected] Publications & Citations: http://scholar.google.com/citations?user=xiL1lscAAAAJ Homepage Salk: http://www.salk.edu/faculty/noel.html Homepage HHMI: http://hhmi.org/research/investigators/noel.html ______________________________________________________________________________________ Begin forwarded message:

From: Joseph Noel Subject: MR-SAD, Yb, combined phases Date: December 19, 2013 at 11:09:06 AM PST To: "[email protected] mailing list"

Hi All,

I am carrying out a structure refinement using experimental phases combined with an MR solution. The experimental phases are being determined from bound Yb3+ cations and the data is collected at one of the Yb absorption peaks of 1.385 A. I do have a few questions about the heavy atom arrangement and then refinement.

At this wavelength, I am also seeing peaks after running MR-SAD that are actually not the Yb cations but the sulfurs on several of the proteins Cys and Met residues. Should I try and change this "heavy atom arrangement" to include Yb and S before determining the final refined phases that I would like to then use as restraints during the structure refinement? If so, its not clear how to do this in Phenix without it finding additional sites that it attributes to Yb but which are actually sulfurs. For MR, this is an exact structure. All I am doing is using Yb which substitute for Mg coordination sites as a source of experimental phases to see how it improves refinement and maps.

Second, when I do refine using the MLHL target function, I am finding quite a bit of excess positive density around the Yb sites. How do I try and account for this? I have attempted to take care of it with ADP and occupancy refinement but that doesn't seem to be enough. The resolution is 2.2 A and the anomalous signal is quite good (alas that is why I am also seeing the sulfurs clearly in the anomalous difference map).

Do I need to change the scattering table since the structure now includes at least 4 Yb cations per asymmetric unit (one 550 amino acid residue protein per asymmetric unit - 68% solvent)? Is it important to refine the anomalous groups? For the latter, one can't refine with phase restraints (MLHL) and anomalous groups together. I have to choose one or the other - no phase restraints and anomalous groups refined, or experimental phase restraints and no anomalous group refinement.

My goal is to use additional experimental measurements - amplitudes and phases to improve refinement, map interpretation and ultimately ensemble refinement.

Any help, comments, criticisms, etc will be very appreciated!

Happy Holidays!

Joe ______________________________________________________________________________________ Joseph P. Noel, Ph.D. Arthur and Julie Woodrow Chair Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA

Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: [email protected]

Publications & Citations: http://scholar.google.com/citations?user=xiL1lscAAAAJ

Homepage Salk: http://www.salk.edu/faculty/noel.html Homepage HHMI: http://hhmi.org/research/investigators/noel.html ______________________________________________________________________________________

Hi Joe,

Second, when I do refine using the MLHL target function, I am finding quite a bit of excess positive density around the Yb sites. How do I try and account for this? I have attempted to take care of it with ADP and occupancy refinement but that doesn't seem to be enough. The resolution is 2.2 A and the anomalous signal is quite good (alas that is why I am also seeing the sulfurs clearly in the anomalous difference map).

see pages 8-14 here: http://www.phenix-online.org/presentations/faq.pdf Please let me know if it doesn't help! Pavel