Hi Steven, Thank you for the information and guidance. When you search for all the ensembles with Phaser do you use “AND” or “OR” searching?
Cheers, Scott While Phil Jeffrey attributed to me the “trick” of aligning the hinge axis of an Fab along the Z direction, I, in turn, must give credit to Mirek Cygler, who explained this to me at the Diffraction Methods in Molecular Biology [now Structural Biology] Gordon Research Conference in 1986. To Scott’s query about searching for “1 domain sequentially and then other domains OR searching for multiple domains all at once?” Inevitably a program like PHASER searches for domains (“ensembles” in its terminology) sequentially. However, as to the practical question of whether to “feed” PHASER all of the domains in one run, that is certainly how I start and it is usually (almost always?) successful. In fact, while I no longer bother to align the hinge axis of an Fab along the Z axis, I now break Fabs into three parts: CL:CH1, VH, and VL to allow molecular replacement to accommodate the “tilt” angle between VH and VL (tilt angle is a term I learned from Gary Gilliland’s talk at the Diffraction Methods in Structural Biology GRC in 2014). This also allows me to search for the highest identity VL and, separately, VH in the PDB to use as probe models. N.B. since I’m usually studying antigen/Fab complex, I’m usually searching for 4 ensembles in one PHASER run: CL:CH1, antigen, VH and VL. ************************************************Scott T. R. Walsh, PhD Assistant Professor University of Maryland IBBR/CBMG 3127E CARB-2 9600 Gudelsky Drive Rockville, MD 20850 USA phone: (240) 314-6478 fax: (240) 314-6225 email: swals...@umd.edu<mailto:swals...@umd.edu>
