Procedures for protein NMR structure calculation by Cyana

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 :=                    # 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
# —- structure calculation —-
read seq $name.seq                      # re-read sequence to initialize
read upl $name.upl                        # read upper distance limits
read 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
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.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’)

Edited on 4/17/2015

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