Documentation and Tutorials

Tutorials
Exercise 1:  How to prepare an unglycosylated FSH dimer for future studies.

1.    load in 4MQW.pdb  (this can be downloaded from the 4MQW entry at the Protein Databank)
        [Console Window]  File  >>  Open...
        [Open]  You may need to navigate to the proper directory to find the appropriate files
        [Open]  select '4MQW.pdb'  >>  {Open}
2.    The resulting structure is a trimer of FSH(dimer)-FSHR complexes.  For modeling purposes, you probably only want a single FSH(dimer)-FSHR, or perhaps only a single FSH dimer.  To pare down to this:
        [Graphics Window]  beside the '4MQW', click {A}  >>  'remove waters'
        [Console Window]  Mouse  >>  Selection Mode  >>  Chains
        [Console Window]  Display  >>  Sequence
        Note that the graphics window now display the single-letter sequence at the top, as well as a scroll-bar.  We will use this sequence representation to find and delete all chains except the A and B chains (which collectively are a single FSH-alpha/FSH-beta dimer)
        [Graphics Window]  You can tell from the "/4MQW//A" characters in white font above the single-letter sequence that the first chain is the A chain.  Please click on any residue letter (green font) within this A sequence, and note that this selects the whole A chain.
        [Graphics Window]  use the scroll bar to scan rightwards on the chain, until you see the white font characters "/B/" which indicate the start of the B chain.  Click on any residue letter (green font) within this B sequence to select the whole B chain.
3.    The two sequences selected in the previous step are what we want to focus on:  a single FSH-dimer.  We are going to extract the dimer and do some final edits.
        [Graphics Window]  beside the '(sele)', click {A}  >>  'extract object'
        [Graphics Window]  to get rid of everything else, beside the '4MQW', click {A}  >>  'delete object'
4.    We do not need the two ethylene glycol molecules, so we will delete these:
        [Console Window]  Mouse  >>  Selection Mode  >>  Residues
        [Graphics Window]  use the scroll bar to scan rightwards on the chain, until you see the green font characters "EDO".  There should be two of the to the far right.  Click on both of them.
        [Graphics Window]  beside the '(sele)', click {A}  >>  'remove atoms'
5.    Lets now add H's to the remaining portion of the protein:
        [Graphics Window]  beside the "all" object, click {A}  >>  'hydrogens'  >>  'add'
6.    Let's now rename and save this molecule:
        [Graphics Window]  beside the "obj01" object, click {A}  >>  'rename object'
        [Graphics Window]  backspace over "obj01" to instead read "fsh"  >>  {enter}
        [Console Window]  File  >>  Save Molecule...
        [Save]  make certain that 'fsh" is highlighted  >>  {OK}
        [Save As]  the default type (*.pdb) and name (fsh.pdb) should be fine  >>  {Save}
7.    Finally we will create a de-glycosylated version for future modeling
        [Graphics Window]  use the scroll bar to scan rightwards on the chain.  Each time you see the green font characters " NAG " click on it (NOTE:  only when there are spaces around the "NAG" because we don't want to delete any Asn-Ala-Gly tripeptides).
        [Graphics Window]  beside the '(sele)', click {A}  >>  'extract object'
        NOTE:  the structure is current form has hypovalent asparagines (i.e., missing one H) at each place we deleted a NAG.  Repeat step 5 in order to replenish H's.
        [Graphics Window]  beside the "fsh" object, click {A}  >>  'rename object'
        [Graphics Window]  type a single "x" at the end of "fsh" to to read "fshx"  >>  {enter}
        [Console Window]  File  >>  Save Molecule...
        [Save]  make certain that 'fshx' is highlighted  >>  {OK}
        [Save As]  the default type (*.pdb) and name (fshx.pdb) should be fine  >>  {Save}
8.    Finally we will use one of the NAG (GlcNAc) residues left over from the previous steps to serve as a carbohydrate fragment template.
        [Graphics Window]  beside the "fshx" object, click {A}  >>  'delete object'
        [Graphics Window]  we can select any one of the four GlcNAc residues, either by clicking on the "NAG" text in the sequence listing, or else by clicking on the structure itself.
        [Graphics Window]  beside the '(sele)', click {A}  >>  'extract object'
        [Graphics Window]  we can now delete 'obj01' by clicking {A}  >>  'delete object'
        Make certain that obj01 is fully protonated by redoing step 5.
        [Graphics Window]  beside the "obj02" object, click {A}  >>  'rename object'
        [Graphics Window]  backspace over "obj02" to instead read "GlcNAc"  >>  {enter}
        [Console Window]  File  >>  Save Molecule...
        [Save]  make certain that 'GlcNAc" is highlighted  >>  {OK}
        [Save As]  change the "Save as type:' to "PKL File (*.pkl)"  >>  {Save}



Exercise 2:  How to prepare a GalNAc residue, based on a GlcNAc template

1.    load in GlcNAc.pkl
        [Console Window]  File  >>  Open...
        [Open]  change 'All Readable' to 'All Files'
        [Open]  select 'GlcNAc.pkl'  >>  {Open}

2.    We are now going to edit GlcNAc to turn it into GalNAc

        [Console Window]  {Builder}
        Look at the arrow mark "1" on Fig.1.  We are going to invert the stereochemistry around this center by swapping the proton and hydroxyl groups, as follows:
        [Console Window]  {Invert}
        [Graphics Window]  click on the chiral carbon as the origin atom
        [Graphics Window]  click on either of the two adjacent ring carbons as the first stationary atom
        [Graphics Window]  click on the other adjacent ring carbon as the second stationary atom
        [Graphics Window]  you will now see that the stereochemistry has been inverted, and there is a prompt for a new stereocenter to be modified.  If we had others to modify we could have done so, but as it is, we can now exit this stereochemical inversion mode as follows:
        [Graphics Window]  {Done}
3.    Let's now rename and save this molecule:
        [Graphics Window]  beside the "GlcNAc" object, click {A}  >>  'rename object'
        [Graphics Window]  backspace over "GlcNAc" to instead read "GalNAc"  >>  {enter}
        [Console Window]  File  >>  Save Molecule...
        [Save]  make certain that 'GalNAc" is highlighted  >>  {OK}
        [Save As]  change the "Save as type:' to "PKL File (*.pkl)"  >>  {Save}
4.    For future reference, we need to record the Atom ID number that will be used as a base for connecting this fragment to proteins or other glycans:
        [Graphics Window]  beside the "GlcNAc" object, click {L}  >>  'atom identifiers'  >>  'ID'
        If your PyMol version is working properly, each atom should now have an ID number listed on screen.  If so, record the number of the atom corresponding to the proton being pointed at by arrow 2 in Fig.1.
        Your PyMol version may have a bug that prevents this label from showing.  If so, you can temporarily force it as follows:
        [Console Window]  {Ray}
        From this, you should see that your connection atom is "15" (as opposed to GlcNAc, which would be "28").  This means that when you connect GalNAc to a protein, the command will be:
            editor.attach_fragment('pk1','GalNAc',15,0)
        Please keep this in mind for later exercises.
5.    To use GlcNAc and GalNAc for future exercises, you will need to copy them to the appropriate fragment folder.  On a typical Windows workstation, this might be something like:
            C:\Python27\Lib\site-packages\pymol\pymol_path\data\chempy\fragments or
            C:\Python27\PyMOL\data\chempy\fragments



Exercise 3:  How to prepare a D-mannopyranose residue, based on a GlcNAc template

1.    load in GlcNAc.pkl
        [Console Window]  File  >>  Open...
        [Open]  change 'All Readable' to 'All Files'
        [Open]  select 'GlcNAc.pkl'  >>  {Open}
2.    We are now going to edit GlcNAc to turn it into GalNAc, first by deleting the acetamide.
        [Console Window]  {Builder}
        Look at the arrow mark "1" on Fig2.tif.  We need to delete this acetamide and position a hydroxyl on the adjacent proton
        [Console Window]  to the right of the "Atoms" label, hit the {Delete} button.
        [Graphics Window]  click on each of the acetamide atoms to be deleted.  Once you have removed the entire acetamide, you will left with a ring carbon that is a bare methylene.
        [Graphics Window]  click the {Done} button.
3.    The next editing step is to turn the axial proton on the newly-bare methylene to a hydroxyl.
        [Console Window]  to the right of the "Chemical" button, hit the {O} button.
        [Graphics Window]  click on the axial proton to be converted.
        [Graphics Window]  click the {Done} button.
4.    Let's now rename and save this molecule:
        [Graphics Window]  beside the "GlcNAc" object, click {A}  >>  'rename object'
        [Graphics Window]  backspace over "GlcNAc" to instead read "d-mann"  >>  {enter}
        [Console Window]  File  >>  Save Molecule...
        [Save]  make certain that 'd-mann" is highlighted  >>  {OK}
        [Save As]  change the "Save as type:' to "PKL File (*.pkl)"  >>  {Save}
5.    For future reference, we need to record the Atom ID number that will be used as a base for connecting this fragment to proteins or other glycans:
        [Graphics Window]  beside the "d-mann" object, click {L}  >>  'atom identifiers'  >>  'ID'
        If your PyMol version is working properly, each atom should now have an ID number listed on screen.  If so, record the number of the atom corresponding to the proton being pointed at by arrow 2 in Fig2.tif.
        Your PyMol version may have a bug that prevents this label from showing.  If so, you can temporarily force it as follows:
        [Console Window]  {Ray}
        From this, you should see that for alpha-mannopyranose, your connection atom would be the axial proton (in my structure this is number "12") whereas for beta-mannopyranose the connector is the equatorial (number "21").  This means that when you connect mannopyranose to a protein, the commands will be:
            alpha:  editor.attach_fragment('pk1','d-mann',12,0)
            beta:  editor.attach_fragment('pk1','d-mann',21,0)
        Please keep this in mind for later exercises.
6.    As before, you will need to copy d-mann.pkl to the appropriate fragment folder.  On a typical Windows workstation, this might be something like:
            C:\Python27\Lib\site-packages\pymol\pymol_path\data\chempy\fragments   or
            C:\Python27\PyMOL\data\chempy\fragments



Exercise 4:  Demonstrate how to glycosylate an unglycosylated FSH dimer by adding a simple Glycan (- GlcNAc - alpha-D-mannopyranose) to Asp 52.

1.    load in fshx.pdb
        [Console Window]  File  >>  Open...
               [Open]  You may need to navigate to the proper directory to find the appropriate files
        [Open]  select 'fshx.pdb'  >>  {Open}
2.    First we change the mouse mode to permit specifying single atoms for modification.
        [Graphics Window]  in the lower right, note the text that says:  "Mouse Mode" and "3-Button Viewing"  >>  click once on the "3-Button Viewing" and it will change to "3-Button Editing"
3.    Now we locate Asp 52.
        [Console Window]  Mouse  >>  Selection Mode  >>  Chains
        [Console Window]  Display  >>  Sequence
        [Graphics Window]  use the scroll bar to scan rightwards on the chain, until you find Asn 52 (it is the "N" in the LVQKN subsequence very close to the "51" residue number marker.  Click on this "N".
        [Graphics Window]  beside the "(sele)" object, click {A}  >>  'center'
        We now clearly see the asparagine that we wish to modify.
4.    Now we select the attachment position for the glycan.
        [Graphics Window]  Asp 52 is centered and highlighted.  Pan in using the right mouse button until you clearly see the asparagine side-chain nitrogen and click on the amide proton that you feel is most solvent exposed.
        [Graphics Window]  Note that in the graphics window, you now have a number of selection sets, including "pk1" which is asparagine proton that will be removed to accommodate the GlcNAc.
5.    We will now add a GlcNAc residue onto the Asp 52 sidechain.
        [Console Window]  In the command line right of the text "PyMOL>", enter the following command:
            editor.attach_fragment('pk1','GlcNAc',28,0)
6.    We may now manually adjust the starting conformation.

Click here to edit text        [Graphics Window]  This is a subjective step and will require some basic instinct, but there is a fair chance that the default GlcNAc orientation relative to the rest of the protein will not be suitable for building a larger glycan.  Fortunately, the circular torsion slider on the protein-glycan connection bond makes for easy torsional editing.
        [Graphics Window]  Holding down the [Ctrl] key, move your mouse pointer onto the recently-added carbohydrate group (directly over an atom or bond), press the left mouse button down and adjust the torsion by moving the mouse.
        [Graphics Window]  adjust the torsion so that the acetamide group doesn't have a bad clash with the protein, and so that the desired attachment for your next carbohydrate group (generally one of the hydroxyl groups approximately opposite to the acetamide) is pointed roughly away from the protein surface.

7.    We can now add the alpha-D-mannopyranose to the GlNAc in a similar manner.
        [Graphics Window]  Click on the hydroxy proton that to which you plan to add the mannopyranose.
        [Graphics Window]  Note that in the graphics window, you now have a number of selection sets, including "pk1" which is GlcNAc proton that will be removed to accommodate the mannopyranose.
8.    We will now add a mannopyranose residue onto the GlcNAc ring.
        [Console Window]  In the command line right of the text "PyMOL>", enter the following command:
            editor.attach_fragment('pk1','d-mann',12,0)
9.    We may now manually adjust the starting conformation in a manner identical to that used in step 6.
        [Graphics Window]  Note that if you wish to modify a torsion any other than the default one, you may choose another bond in 'Three-Button Editing' mouse mode by pointing your mouse to it and going [Ctrl]-(right-click).
10.    Add additional carbohydrate groups as desired (e.g., as per steps 7-9) and save the resulting structure:
        [Graphics Window]  beside the "fshx" object, click {A}  >>  'rename object'
        [Graphics Window]  give the molecule a new name  >>  {enter}
        [Console Window]  File  >>  Save Molecule...
        [Save]  make certain that your recently edited molecule is highlighted  >>  {OK}
        [Save As]  the default type (*.pdb) and name should be fine  >>  {Save}