MethylFLYA example calculation
------------------------------

This directory contains an example MethylFLYA calculation for the automated
structure- and NOESY-based assignment of methyl groups in large proteins.

The example data is for the N-terminal domain of E. coli Enzyme I (EIN; 259 aa).

The complete assignment calculation is performed by first running the RUN.cya
macro that calls PREP.cya to make expected peaks using three different NOE
distance cutoffs, and starts parallel FLYA automated assignment runs with 
CALC.cya for the different NOE distance cutoffs. After completing the FLYA 
runs, consensus chemical shifts are obtained with CONSOL.cya, which must be
run separately.

The preparation macro, PREP.cya, performs the following tasks:
1. The C13HSQC peak list, C13HSQC.peaks, which is assigned to methyls of the 
   correct amino acid type (and arbitrary residue numbers that are not used), 
   is split into amino acid type-specific peak lists, C13HSQC_X.peaks, with 
   X = A, I, L, V.
2. The peaks in the unassigned short mixing-time 4D CCNOESY peak list, 
   HCcCH.peaks (formally treated as an HCCH TOCSY-type experiment),which 
   contains connections between the two methyl groups of Leu or Val, are 
   assigned to amino acid types (and irrelevant, arbitrary residue numbers)
   according to the closest C13HSQC peaks, and the peak list is split into 
   amino acid type-specific peak lists, HCcCH_L.peaks and HCcCH_V.peaks.
3. The unassigned 4D NOESY peak list, CCNOESY.peaks, is treated similarly,
   and split into amino acid type-specific peak lists, CCNOESY_XY.peaks,
   with X, Y = A, I, L, V.
4. The macro peaklists.cya that specifies the generation of expected peaks
   during the FLYA calculations is written. It will be executed in CALC.cya.

Input files:

demo.seq        sequence
demo.pdb	3D structure
C13HSQC.peaks	2D C13HSQC peak list with amino acid type assignments
                (peaks are assigned to methyl groups of the correct
		amino acid type but arbitrary residue number)
HCcCH.peaks     short mixing-time 4D CCNOESY for intraresidual Leu/Val connections  
CCNOESY.peaks	4D CCNOESY peak list, unassigned
ref.prot	reference chemical shifts (for comparison only)
init.cya	initialization macro
RUN.cya		automated assignment calculation (calls PREP.cya, CALC.cya)
PREP.cya	prepare peak lists for FLYA
CALC.cya	run FLYA assignment calculation
CONSOL.cya	determine consensus chemical shifts

Main output files:

consol.prot	consensus chemical shifts
consol-strong.prot  strong (confident) consensus chemical shifts
consol.tab	table of consensus methyl assignments
consol.pdf	plot of consensus methyl assignments
demo_d4.5	directory for FLYA calculation with NOE distance cutoff 4.5 A
demo_d5.0	directory for FLYA calculation with NOE distance cutoff 5.0 A
demo_d5.5	directory for FLYA calculation with NOE distance cutoff 5.5 A

Ref.: Pritisanac et al. https://doi.org/10.1101/538272 (2019). (MethylFLYA)
      Venditi et al. J. Biomol. NMR 51, 319-328 (2011). (EIN)

