Exercise 5.
Build a loop

1. Load structure 3GAR of enzyme GART (GlycinAmide Ribonucleotide Transformulase) - this is a high pH monomeric structure with E70A mutation and without substrate
   Re-load structure 3GAR, select it in the Header of Control Panel, and rename it to 3GAR_loop using Tool Bar Edit Rename current layer
   Hide this structure
   Display residues His119-Asn127
   Note that middle residues Thr120-Glu126 form a helix
   Our goal is to replace this helix with a loop
2. Loop building
   In Tool bar Build Build loop
   You will be asked to select 2 anchor residues - select the 2 residues immediately outside the helix, His119 at one end and Asn127 at the other end - this is for the energy minimization that will follow the loop building
   This calculation will take a while because the program scans a loop data base - be patient!
   When done a list of several loops is shown
   At the Header there is a scoring system classification based on van der Waals clashes, potential and force field energy
   Then, follows a long list with deviations from ideal C--N+ bond length and ideal Ca--C--N+ and C--N+--Ca+
   By clicking each row of the list you select a new loop that is now displayed in the Main Window
   Note that some calculations of the above mentioned deviations are done on the fly
3. Loop selection
   structures 3GAR and 3GAR_loop are superimposed, make sure that only the regions His119-Asn127 are displayed
   Also, display the ribbons of these segments
   By now you must know that using Tool bar's Display Use openGL rendering and Render in solid 3d you get better looking solid ribbons
   Color backbone-side chains and ribbons in white for 3GAR and in yellow for 3GAR_loop
   Rotate the structures in a helical wheel representation for better view of the differences between helix and loop
   Scan now the loop lists to visualize the different loops
   Select a loop that you like
   Save layer as 3GAR_loop.pdb
   Remove residues 1-110 and 132-end and save layer as 3GAR_looponly.pdb
4. pH-dependent helix-coil transition
   Now, load structure 2GAR - (GlycinAmide Ribonucleotide Transformulase) - this is a low pH monomeric structure with E70A mutation and without substrate
   Try to select the segment His119-Asn127 - it is missing! - indeed the segment 110-131 is missing
   When are coordinates missing in crystal structures?
   Answer: Lack of electron density caused by high structural mobility
   This is the case of a pH-dependent helix-coil transition
   Our goal is to transfer the generated loop coordinates using the structure 3GAR_loop to the structure of 2GAR
5. Loop transfer
   Close structure 3GAR
   In 3GAR_loop select residues 1-110 and 132-209 ONLY
   Select all residues in 2GAR
   Fit the 2 structures using "selected residues" option (not magic fit)
   Save 2GAR as 2GAR_fit
   In 3GAR_loop select residues 111-131 ONLY
   Select all residues in 3GAR_looponly
   Fit the 2 structures using "selected residues" option (not magic fit)
   Save 3GAR_looponly as 3GAR_looponly_fit.pdb
   Save 2GAR_fit as 2GAR_fit_a.pdb
   Save 2GAR_fit as 2GAR_fit_a.pdb
   Close all structures
   Load in the following order structures: 2GAR_fit_a.pdb, 3GAR_looponly.pdb, 2GAR_fit_b.pdb
   In Tool Bar Edit Create merged layer from selection - this will create a new layer named _merge_
   In Tool Bar Edit Rename current layer - this will accomplish re-numbering the residues in the correct order
   Save _merge_ as 2GAR_loop.pdb
6. Energy minimization for the re-constructed loop
   Re-name _merge_ to 2GAR_loop_Eminimized
   Select residues 110-132 - remember 111-131 is the transferred loop and 110, 132 are the anchor residues to the remaining of the backbone
   I recommend hiding all other structures and showing only residues 110-131 of layer 2GAR_loop_Eminimized
   Do energy minimization
   Save layer as 2GAR_loop_Eminimized.pdb
7. Structural comparison
   Close all structures and load only 2GAR_loop.pdb and 2GAR_loop_Eminimized.pdb
   Hide all residues except 110-131 for both structures
   Check the effect of energy minimization on some side chains
   Close structure 2GAR_loop.pdb
   Load structure 3GAR.pdb
   Magic fit 3GAR.pdb and 2GAR_loop_Eminimized.pdb if necessary
   Draw ribbons in different colors
   Focus on segment 110-132
   Rotate structures to a helical wheel orientation
   Enjoy the result!