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


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

Ref.: Pritisanac et al. Nat. Commun. 10, 12 (2019).


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. In this demo, these are the 
		correct residue numbers; in general, residue numbers only have 
		to match the correct residue type, and, if geminal correlations
		in Leu/Val are to be used, be equal for the two methyl groups
		of a Leu/Val residue.
CCNOESY3D.peaks	3D CCNOESY peak list, unassigned
ref.prot	reference chemical shifts (optional; used only for comparison) 
init.cya	initialization macro; defines labeling pattern and shift tolerances
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

The example data is for the Hbeta2m protein (99 aa), as described in
Nerli et al. Nat. Commun. 12, 691 (2021). 
Original data from http://methylassignment.chemistry.ucsc.edu/io.


Procedure:

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.

A complete methyl assignment calculation thus comprises the following steps:

1. Prepare a file with the sequence file of the protein in CYANA format.
   (here: demo.seq)

2. Prepare the 2D C13HSQC peak list in XEASY format, with pseudo assignments
   to residues of the correct type.
   (here: C13HSQC.peaks)
   
3. Prepare the (unassigned) 3D or 4D NOESY peak list in XEASY format.
   (here: CCNOESY3D.peaks)

4. Edit the initialization macro, init.cya, to set the variable 'name' 
   that is used for the sequence file and some output files, and to specify
   methyl-labeled residue types and the chemical shift matching tolerances.
   
   
5. Edit the parameters section in the PREP.cya macro to specify the names
   of the input structure and peak list files.
   
6. Execute the command 'cyana RUN' to prepare and run FLYA methyl assignment
   with three different NOE distancce cutoffs (specified in RUN.cya). In 
   total, 300 individual FLYA assignments will be performed, which can take
   a substantial amount of time.
   
7. Execute the command 'cyana CONSOL' to consolidate the results of the
   300 individual FLYA calculations into a single consensus assignment of
   the methyl groups.


Preparation macro details:

The preparation macro, PREP.cya, creates three subdirectories demo_d4.5, 
demo_d5.0, demo_d5.5 for the three NOE distance cutoffs and performs the 
following tasks in each of these subdirectories:

1. Hydrogen atoms are attached to the input 3D structure, if necessary. The
   input structure can be a single (e.g. X-ray) structure, or a bundle of 
   conformers.
   --> ref.pdb
   
2. 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.
   
3. The peaks in the unassigned 3D NOESY peak list, CCNOESY3D.peaks, 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,
   --> CCNOESY3D_X_Y.peaks, with X, Y = A, I, L, V.
   
4. A 4D peak list, HCcCH.peaks (formally treated as an HCCH TOCSY-type experiment),
   is prepared based on the (pseudo) assignments in the C13HSQC peak list to specify 
   intraresidual connections between the two methyl groups of Leu or Val, and split 
   into amino acid type-specific peak lists. A geminal correlation is generated for
   each Leu or Val residue to which exactly two peaks are assigned in the C13HSQC
   peak list (the residue numbers of the two 2D peaks must be equal and refer to an
   existing Leu or Val residue but are otherwise arbitrary).
   This optional step is skipped if the variable 'hccch' in the parameters section is 
   empty.
   --> HCcCH_X.peaks, with X = L, V
   
5. The macro peaklists.cya that specifies the generation of expected peaks
   during the FLYA calculations is written. It will be executed in CALC.cya.
   --> peaklists.cya


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
