Download it from https://github.com/dgaston/FunDi ⇒ ZIP button

Install it with these commands:

unzip dgaston-FunDi-229c4d6.zip
cd dgaston-FunDi-229c4d6/
unzip qmmraxml.zip
export PATH=$PATH:qmmraxml  # Or mv qmmraxml/qmmraxmlHPC to a PATH folder

if not working, you may need to recompile qmmraxmlHPC

unzip dgaston-FunDi-229c4d6.zip
cd dgaston-FunDi-229c4d6/
unzip qmmraxml.zip
cd qmmraxml
make  clean   # remove all files
make          # compile qmmraxmlHPC
cd ..
export PATH=$PATH:qmmraxml  # or move  qmmraxmlHPC in a path folder.

FunDi needs PERL release 5.10 or higher (maybe with threads enabled).

perl FunDi.pl -a GAPDH_alignment.phy -o gapdh_subtree -m LG -s gapdh_subtree.def -P qmmraxml -r 4 -t gapdh.tre

(It can take time)

Parameters:

• -a: multiple alignment file in PHYLIP format.
• -o: basename for output.
• -m: Substitution matrix (qmmraxml ⇒ LG).
• -s: definition of groups, one line = one subgroup.
• -P: phylogenetic tool to estimate parameters.
• -r: number of categories for the gamma distribution.
• -t: phylogenetic tree in NEWiCK format.

More details with the help:

perl FunDi.pl -h

The most important file is “FunDi_Posterior_Scores.txt”.
You can load it in your spreadsheet (tab separated, activate special detection of numbers).
The numbering of sites starts from 0.
Sites with a posterior probality P(FD) with 0.50 or more could be interesting.
In particular:

• site 38 (= 39 in Jalview) (= 34 in the structure 2PKQ)
• site 193 (=194 in Jalview) (=188 in the structure 2PKQ)
• site 195 (=196 in Jalview) (=191 in the structure 2PKQ)

Open Jalview, load the file GAPDH_alignment.phy, remove the first line and visualisation the sites.

Open PyMol and load protein 2PKQ

Plugins-> PDB Loader Service 

Change the visualisation mode

all->S->Show->as->Cartoon
all->C->Color by->chain

In the PyMol command line, select the sites of interest…

select ImportantSites, resi 34+188+191 

… and change the visualisation mode:

(ImportantSites)->S->Show->Spheres
(ImportantSites)->C->Color by->chain