need to transfer prok.pir m230d_2008_scaled.mtz (if not already present) ccp4i & cad load m230d_2008y_dm.mtz all columns load m230d_2008_scaled.mtz FP_pmsf SIGFP_pmsf FreeR_flag output m230d_2008y_dm1_freer.mtz check off Complete reflection list and extend freeR column FreeR_flag arp/warp MTZ in m230d_2008y_dm1_freer.mtz Fobs FP_native PHIB PHIDM 280 residues in 1 molecule 3 cycles atuobuilding 15 total cycles Use FreeRflag takes 19 minutes 30 seconds refmac5 MTZin m230d_2008y_dm1_freer.mtz PDBin your model weight=0.3 OK check output R-factor give out prizes give talk check log from arp/warp view coordinates with pymol cp 20_warpNtrace.pdb prok_pmsf_refmac0.pdb delete waters and refine PMSF. Refmac5 MTZin m230d_2008y_dm1_freer.mtz PDBin prok_pmsf_refmac0.pdb weight=0.3 OK check output R-factor view with coot peak 1 sulfate peak 2 n-term peak 8 cis peptide residue 174 build from 174 to 172 peak 37 PMS at Ser225 file->get monomer->pms calculate->merge molecule Last week you asked Arp/wARP to automatically trace the electron density map you calculated for proteinase K. The Rwork=15.1%, Rfree=19.2%. This model is fairly complete except for some odds and ends. Today you will be using this arp model to begin refinement of the prok-pmsf complex. PMSF is the inhibitor which covalently bonds to the active site serine of PROK. We begin by calculating a difference Fourier map in order to see what differences exist between the two structures. Hopefully there will only be a few changes. The primary change should be the addition of PMSF. Fobs(PMSF)-Fcalc(native) phi(native) This only works if the structures are isomorphous. If the two data sets had very different unit cells then this method would not work so well. We will get positive and negative peaks in this map. Positive peaks in this map should correspond to atoms in the PMSF complex that were absent in the native protein. Negative peaks in this map should correspond to atoms in the native complex that are absent in the PMSF complex You will alter the arp/warp model to make it fit the difference maps. Start with the highest features first. For example: 1) flip a tryptophan ring, 2) build a cis peptide that was absent 3) build in a PMSF inhibitor. 4) there will be lots of ordered water molecules that you will not be modeling on this round. When you are done modifying the coordinate set, you will write out coordinates. This will be your first model representing the ProK-PMSF complex. You submit the coordinates to refinement against the PROK-PMSF structure factors. The refinement program, refmac, will move the xyz coordinates and adjust temperature factors (4 parameters for each atom) to minimize the discrepancy between Fobs(Prok-PMSF) and Fcalc(Prok-PMSF). Hence, the working Rfactor will decrease. Hopefully the Free Rfactor will also decrease. A new difference Fourier map is calculated Fobs-Fcalc, that will hopefully have less features than the previous difference map. If there is time, we can check this map. We will be reporting the statistics of the refinement in a table, which you will hand in at the end of class. In addition to statistics regarding structure factors (# of reflections used, Rwork, Rfree, etc). You will be reporting statistics regarding the quality of the geometry of the model. RMS deviations from ideal bond lengths bond angles and # of outliers from the Ramachandran plot. The statistics you get will be rather good because of the quality of the data. You can use these a standard when judging other papers. In addition, there are two other structure validation programs that you can use to judge the quality of the model. They analyze parameters that are not normally restrained in the refinement procedure. They look at atom environements and how representative these environments correlate from a library of atom environements derived from a collection of high qualtity structures. Mike