[phenixbb] ligand possibly bound to active site cysteine

Jorge Iulek iulek at uepg.br
Thu Jul 9 03:27:00 PDT 2020

Dear all,

     I am refining a structure of a Glyceraldehyde 3-phosphate 
dehydrogenase (GAPDH) (converts glyceraldehyde 3-phosphate into 
D-glycerate 1,3-bisphosphate) , 
https://www.brenda-enzymes.org/enzyme.php?ecno= .

     It turns out that its active center cysteine presents bound ligands 
, covalently or not to be determined if possible (data resolution 2.51 A).

     I would like to get help on two issues, (1) what the ligand might 
be and (2) how to treat it (correct me) in phenix.refine.

1) The protein was expressed in E. coli; it had much contact with 
glycerol and crystallization conditions include the 
"ethylene-glycols-mix" ("a mixture of diethylene glycol, triethylene 
glycol, tetraethylene glycol, and pentaethylene glycol"). Nevertheless, 
no NAD cofactor was added, and there is no electron density for it. 
Otherwise, phosphate was also present in crystallization condition.

In a previous study, I learned that glycerol might also contain minor 
amounts of ethylene glycol. I wonder, nevertheless, about glyceraldehyde 
(and note resemblance with the substrate).

Catalytic mechanism includes a hemithioacetal intermediate 
) such that cysteine SD is bound covalently to a carbon. I wonder also 
how much this might attack an ethylene glycol and their likes.

Pictures for the density are shown at for the 4 monomers of the a. u., 
first 4 photos: https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA (blue 1 sig 
for e. d. maps, green 3 sig for Fourier difference maps) . Density is  
different among them to different degrees. The nearby threonine, in some 
cases, seems to interact with a blob (and it is helped by other 
threonine and a serine) which + - might accommodate a phosphate.

I have tried to fit a number of molecules, e.g., the substrate (but not 
really good in all monomers for the phosphate moiety), glycerol, 
ethylene glycol and its di and tri (found also in other places in the 
structure) and now I went for  glyceraldehyde (though, I have doubts 
that there is other - apart from the one eventually bound to S - 
tertiary carbon). Apart from the difficulties on searching for the best 
fitting molecule (and consider their intrinsic flexibility) I do not 
manage to establish distance between them and Cys SD (and there goes the 
second question).

2) I could not devise how to set a proper distance between any of the 
ligands and the Cysteine, be it to check for a covalent bond or to 
establish a van der Waals restriction. I tried:

     bond {
       action = *add delete change
       atom_selection_1 = chain A and resid 153 and name SG
       atom_selection_2 = chain N and resid 5 and name C3
       symmetry_operation = None
       distance_ideal = 1.803
       sigma = 0.1
       slack = None
       limit = -1.0
       top_out = False

     Results are also show for my Glyceraldehyde trial, last 4 photos, 
. Note clashes.

     Curiously , for some of the bonds to be added, I receive the message:

"  Atom "HETATM 9835  O2  3GR N   5 .*.     O  " rejected from bonding 
due to valence issues."

     which seems to point to oxygen atoms, though I declare carbon atoms.

     Helps welcome, thank you.


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