Structure calculation and automated NOESY assignment with CYANA (EMBO 2019)
Automated NOESY assignment and structure calculation
Automated NOE restraint assignment and the structure calculation by torsion angle dynamics is included in 'CALCstructcalc.cya' (see above).
Alternatively, you can also perform automated NOE restraint assignment and the structure calculation separately with the 'CALCauto.cya' macro. The 'flya.prot' file from the automated resonance assignment (e.g. with 'CALC.cya'; backbone and side-chain assignments are required!) will be used together with the (unassigned) NOESY peak lists to assign the NOESY peaks and to generate distance restraints in order to compute the three-dimensional structure of the protein.
TALOS-N can be used to generate torsion angle restraints from the backbone chemical shifts in 'flya.prot'. To do this, use the CYANA commands
read flya.prot talos write talos.aco
This will call the program TALOS-N and store the resulting torsion angle restraints in the file 'talos.aco'.
For further information about automated NOESY assignment you can consult the Tutorial Structure calculation with automated NOESY assignment (which uses different file names than we have here).
To speed up the calculation, you can set in 'CALCauto.cya':
structures:=50,10 steps=5000
These commands tell the program to calculate, in each cycle, 50 conformers, and to analyze the best 10 of them. 5000 torsion angle dynamics steps will be applied per conformer.
Seven cycles of automated NOE assignment and structure calculation will be performed by running the command
cyana "nproc=25; CALC"
Statistics on the NOE assignment and the structure calculation can be obtained with the command 'cyanatable -l'.
The final structure will be 'final.pdb'. The optimal residue range for superposition can be found with the command
cyana overlay final.pdb
or with the CYRANGE web server.
You can visualize the structure bundle, for example, with PyMOL. To superimpose the structures in PyMOL, you can use the internal PYMOL command with the appropriate residue range:
intra_fit resi 8-110 set all_states