1. Use nmrviewJ (or CARA, CCPN-analysis, or Sparky, etc.) to finish protein backbone assignments with spectra including 2D N15 HSQC, 3D hncacb and cbcaconh, hnca and hncoca, hnco and hncaco , and further finish all side chain assignments with spectra including cconh, hcconh, hcch-tocsy and hcch-cosy.
2. Here suggest to use CCPN-analysis to do two (N15 and C13 edited) 3D NOESY spectra analysis, save peak files and proton chemical shift files as the xeasy or cyana format in order to use conveniently in the next CYANA calculation steps.
3. Prepare necessary input files including seq_file, n15_peaks_file, c13_peaks_file, n15_prot_cs_file, and c13_prot_cs_file from CCPN-analysis and dihedral_angal_file from Talos for CYANA NOE automatic assign calculation (run CALC.dya).CALC.dya looks like the following: peaks := c13.peaks,n15.peaks # names of NOESY peak lists prot := cupa # names of chemical shift lists constraints := cupa.aco # additional (non-NOE) constraints tolerance := 0.040,0.030,0.45 # chemical shift tolerances calibration := # NOE calibration parameters structures := 100,20 # number of initial, final structures steps := 10000 # number of torsion angle dynamics steps rmsdrange := 33..121 # residue range for RMSD calculation randomseed := 434726 # random number generator seed noeassign peaks=$peaks prot=$prot autoaco
4. Load the final output n15-cycle7_peaks and c13-cycle7_peaks files into the two 3D NOESY spectra, and do peak correction manually, and save the new peaks files and proton chemical shift files as the new input files, and further do CYANA structure calculation (run anneal.dya).anneal.cya looks like the following: peaks := c13.peaks,n15.peaks # names of peak lists prot := cupa-final # names of proton lists tolerance := 0.040,0.030,0.45 # chemical shift tolerances # order: 1H(a), 1H(b), 13C/15N(b), 13C/15N(a) calibration:= # calibration constants (will be determined # automatically, if commented out) dref := 4.2 # average upper distance limit for # automatic calibration
if (master) then # —- check consistency of peak and chemical shift lists—- peakcheck peaks=$peaks prot=$prot # —- calibration —- calibration prot=$prot peaks=$peaks constant=$calibration dref=$dref peaks calibrate “**” simple write upl $name-in.upl distance modify write upl $name.upl end if synchronize # —- structure calculation —- read seq $name.seq # re-read sequence to initialize read upl $name.upl # read upper distance limits read aco $name.aco # read angle constraints seed=9925 # random number generator seed calc_all structures=100 command=anneal steps=10000 # calculate 100 conformers overview $name.ovw structures=20 pdb # write overview file and coordinates
5. Format conversion from CYANA to XPLOR/CNS for xplor-nih structure energy minimization refinements (run Cyana2cns.dya).
Cyana2cns.dya looks like the following:# Convert ACOs to CNS/XPLOR translate off read aco cupa.aco translate xplor write aco cupa_dih.tbl xplor # Convert PDB to CNS/XPLOR translate off pseudo=0 # do not include pseudoatoms in PDB read pdb cupa translate xplor write pdb cupa_xplor.pdb all # Convert UPLs to CNS/XPLOR translate off read upl cupa.upl #read upl man.upl translate xplor pseudo=3 # Use Xplor/CNS pseudoatom names (e.g. HB*) write upl cupa_noe.tbl xplor
6. Prepare input files including cupa_dih.tbl, cupa_noe.tbl, and a series pdb files from cupa_xplor.pdb, and run xplor-nih python script to refine the structures:
xplor –py refine_accept_test.py > refine_accept_test.out
Edit the input files in the next lines in the python script:… numberOfStructures=20 if quick else 20 … inTemplate=”model_0″ + “*.pdb” … noe = create_NOEPot(‘noe’, ‘cupa_noe.tbl’) … torsionFile=’cupa_dih.tbl’ …
Edited on 4/17/2015