Pymol–  Tutorial    Exercise  1:  Introduction    “File  –>  Open”  3OG7      Save  your  session  by  doing  “File  –>  Save  Session  As...”  (don’t  forget  to  save  your  work  regularly  by  “File  –>  Save  Session”  

 

PyMol  can  open  more  than  one  molecule  at  a  time,  or  separate  complex  PDB  files  into  individual  components.  Each  opened  or  loaded  molecule  is  given  a  name  within  the  “Names  Panel”  (right  part  of  the  window).  The  first  name  is  always  “all.”  Clicking  on  the  name  itself  will  undisplay  the  corresponding  molecule(s)  (temporarily  invisible).  Clicking  again  on  the  name  will  display  the  molecule.  

The  ASHLC  menu    is  abbreviated  for  Action,  Show,  Hide,  Label  and  Color.  

For  example,  for  a  better  overview  “S”“Show  as”“cartoon”.  This  hides  everything  and  shows  the  protein’s  backbone  representation.    Go  to  the  following  menu  cascade:  Setting  >  Cartoon  >  Cylindrical  Helices  Select   this   option   again   to   remove   its   effect   and   do   the   following:   Setting   >  Cartoon  >  Fancy  Helices    PyMol  is  optimized  for  a  3-­‐button  mouse:  

Rotation  around  the  X  or  Y  axis:       (left)  click  and  drag  Rotation  around  the  Z  axis:       (left)  click  on  the  top  left  or  right  corner  Translate  (move  sideways)  X  or  Y:     click  middle  button  and  drag  Zoom  (move  along  Z  axis):     click  right  button  and  drag  up  or  down    Now  start  to  “experiment”  with  your  mouse.    

 Explore  the  “preset”  options  of  the  Action  set  of  the  ASHLC  menus:  -­‐  simple  -­‐  simple  (no  solvent)  -­‐  ball  and  stick  -­‐  b  factor  putty  ….  -­‐  publication          Save  final  images:    “File  –>  Save  Images  As...”  –>  “PNG…”    The  image  will  be  saved  as  a  PNG  image.  However,  this  image  is  rather  crude   in   terms   of   graphics   and   resolution.   PyMol   offers   an   internal  “ray  tracer”  to  create  rendered  images  with  a  high  visual  quality  ideal  for  publication.    To  create  a  standard  ray-­‐traced  image  of  the  current  Viewer  scene,  click  the  “Ray”  button  and  save  the  image  as  PNG.    Type  the  commands  “set  ray_trace_mode,    1”  “set  ray_trace_color,  black”  for  a  black  line.            

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L02 – page 9

2BIW.pdb2 > A > preset > default

This command has a similar effect but is not the

same as the following cascade: hide everything and

show lines:

2BIW.pdb2 > H > everything

and

2BIW.pdb2 > S > lines

Note: the “preset” options will set some variables that

are specific to these views and may change further

drawings. To remove the effect of these presets

affecting an object representation, use the

A>preset>default menu cascade reset parameters.

Note: to get back to the original opening view simply type reset at the PyMOL> line command.

2) Exploring more

Explore the other menus of this series.The cascade menu 2BIW.pdb2 > A > preset is assumed in the following commands:

simple

The tracing can then be made thicker by unselecting thesmooth option with the following menu cascade:Setting > Ribbon > Smooth

Note: a set of 3 histidines is also shown.

ball and stickThis is not very useful for alarge protein such as this.

b factor putty

The segments with the highest temperature factor are shownas thicker cylinders. Regions of better resolution have thinnerdiameter and are usually found at the core of the protein.Mostly loops in the outside of the protein wobble: the coreportions of the proteins usually appear more stable than theexternal loops. This is mostly useful for crystallographers butis a cool representation.

technical

Color domains in separate rainbow colors and shows backbone andside chains.Note that a subset name appears in the Names Panel(2BIW.pdb2_pol_co) that control the dashed-line hydrogen bonds.

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L02 – page 7

6) Final image(s)

If you still see the selection dots over the ligand from the previous section simply clickanywhere on the white background to unselect. Alternatively click on the “Hide-Sele” button atthe top right hand side of the “external GUI.”

Rotate the molecule to find a perspective that you deem instructive of the conformation of theprotein and it’s bound ligand.

Follow this menu cascade to save the image currentlywithin the Viewer:

File > Save Image…

Then replace the default word “pymol” to give a name to

the file you want to save, e.g. image1

The image will be saved as a PNG image on the desktop

However this image is rather crude in terms of graphics and resolution. PyMol offers aninternal “ray tracer” to create stunning rendered images with a high visual quality much morepleasant to the eye and ideal for publication.

To create a standard ray-traced image of the current Viewer

scene, click the “Ray” button at the top right of the “external

GUI.” This will take a few seconds to a few minutes depending on

the complexity of the PDB file and the the chosen display, and

will also depend on the speed of the computer CPU. Once

rendered, the image appears within the Viewer. To save the file,

use the save cascade as above: File > Save Image…

Zoomed side-by-side comparison between the pymol image and the ray-traced image: note the jagginessof the original image and the smooth appearance of the ray-traced image, with shadows as a bonus.

When   you   made   yourself   familiar   with   PyMOL   and   its   mouse  movements,   click   on   the   “S”   in   the   bottom-­‐right   corner.   This  shows  you  the  protein’s  sequence.    At   the  beginning  of   the  protein   sequence,   there   is   a   compound,  which  was  crystallized  together  with  the  structure  of  the  protein.  Select  the  compound  “O32”.    

   You  now  see  a  second  panel  called  “(sele)”  under  “3OG7”  which  represents  the  currently  selected  part  of  the  structure.  Use  it  to  show  the  ligand  “O32”  in  sticks.    

 Next  we  change  the  color  of  the  ligand.    Click   “(sele):C   –>   by   element   –>   and   select   a   color  that  you  like”.    

   Zoom   to   the   ligand   using   your   mouse.   You   can  center  the  ligand  (or  any  other  part  of  the  structure)  by   selecting   it   and   then  pressing   the  mouse  wheel  or  type  “zoom  sele”.        

   

We  are  now  interested  in  the  amino  acids  around  the  ligand  –  the  so-­‐called  “binding  site”.    To  explore  this  further,  select  the  ligand,  click  “(sele):  A  –>  modify  –>  around  –>  residues  within  4  A”.  You   have   now   all   residues   within   4   Angstrom   selected.   Without  clicking   anywhere   else   (this   would   undo   your   selection)   click  “(sele):    S  –>  side  chain  –>  sticks”  and  “(sele):    C  –>  by  element  –>  and  select  a  color  different  than  the  ligand”    

     

       

Your  window  should  look  somewhat  like  this:  

   Now  we  want  to  look  at  an  important  interaction  between  the  ligand  and  the  protein:  The  ligand  O32  is  forming  hydrogen  bonds  interactions  with  the  backbone  of  Gln530  and  Cys532.  Scroll  in  the  sequence  at  the  top  to  find  this  residue  and  select  it.  To   confirm   that   you   have   selected   the   right   residue,   click   “(sele):   L   –>   Residues”.   You   will   find   your  residue  labeled  by  name  and  residue  number.    Change  size  and  font  in  “Setting  >  Label”  “(sele):  S  –>  Sticks”  Furthermore  there  are  two  command  line  options  you  should  know  for  selecting  residues:  “sele  resn  gln”  which  selects  all  aspartic  acids  and  “sele  resi  530”  which  selects  residue  number  530    Next  we  want  to  confirm  that  there  is  a  strong  interaction  between  Lys483,  Gln530  and  Cys532  and  the  ligand  O32.  We  do  this  by  measuring  the  distance.  Therefore  use  the  small  GUI  window,  click  “Wizard  –>  Measurement”   and   then   in   the   main   window   click   the   two   atoms   whose   distance   you   want   to   be  measured.  

 

   Which  atoms  of  O32  and  Gln530  are  interacting  through  a  hydrogen  bond?  What  is  the  distance?    Which  atoms  of  O32  and  Cys532  are  interacting  through  a  hydrogen  bond?  What  is  the  distance?  Which  atoms  of  O32  and  Lys483  are  interacting  through  a  hydrogen  bond?  What  is  the  distance?    Reproduce  (more  or  less)  the  following  pictures,  which  display  amino  acids  interacting  with  the  ligand  O32  

 

 

Select  “Movie  >  Camera  loop  >  Nutate  >  30  deg.  Over  4  sec.”  It  creates  120  states  of  the  figure  …    Click  the  play  button.  “File  >  Save  Movie  As  >  MPEG”  You  can  save  the  movie  with  high  quality  “Ray”  figures.  Click  Ray  and  save  again.  Open  the  file  in  powerpoint.  

   

 

Exercise  2:  Cavities  and  molecular  electrostatic  potential    Open  5P21  

Hide  everything  

Show  cartoon  

Copy  residues  GNP167  and  MG168  to  a  new  object  

Remove  GNP167  and  MG168  

Rename  the  new  object  to  “GNP”  

Set  Surface  Cavities  &  Pockets  (Culled)  

Show  surface  

Show  GNP  as  spheres  

Set  Surface  Exterior  normal  

Hide/Show  GNP  

Calculation  of  Electrostatic  Surface  with  APBS,  via  PyMol  

ABPS  (Adaptive  Poisson-­‐Boltzmann  Solver,  http://www.poissonboltzmann.org/apbs/)  calculates  the  electrostatic  properties  at  the  molecular  surface  by  solving  the  Poisson-­‐Boltzmann  equation  (http://en.wikipedia.org/wiki/Poisson–Boltzmann_equation).  PyMol  has  an  integrated  plugin  that  calculate  ABPS.  

PyMol>Plugin  open  “APBS  tool”  

In  the  “Main”  menu  select  “Use  PyMOL  generated  PQR  and  existing  Hydrogens  and  termini”    

In  the  “Configuration”  menu  select  “Set  grid”  (  the  x,y,z  values  of  the  grid  is  define)  (protein  and  solvent  dielectric  are  defined  for  soluble  proteins,  and  ion    concentration  is  setup  by  default)  

Select  “Run  APBS”  and  wait  ……  until  the  “Visualization”  menu  automatically  shows  up.  

In  the  “Visualization  menu”  select  “Color  by  potential  on  sol.  acc.  surf”  and  select  “Update”  

Change  the  electrostatic  range  value  by  modifying  “Low”  to  -­‐6.0,  “Middle”  to  0.0  and  “High”  to  4.0  and  select  “Update”.  Play  with  these  values.  

Hide/Show  GNP  

Exercise  3:  Superimposition  of  structures  

We  want  to  compare  the  2Y03  structure  with  3PWH.    Open  2Y03  (β1-­‐adrenergic  receptor)  Display  2Y03  as  a  cartoon.  It  is  a  dimer  (contains  chains  A  and  B).  Remove  atoms  of  chain  B    type  “sele  chain  B”  “(sele):  A  –>  remove  atoms”  Be  careful,  PyMOL  has  no  “undo”  function.  Removed  atoms  can’t  be  brought  back!    Open  3PWH  (adenosine  A2A  receptor)  Display  3PWH  as  cartoon.    Now  we   want   to   compare   the   adrenergic   and   the   dopamine   receptor.   You   will   see   that   they   are   in  different  positions  and  comparison  between  them  is  impossible.    Click  “3PWH:  A  –>  align  –>  to  molecule  –>  2Y03”.    In  the  GUI  window  you  can  see  the  rmsd  value  between  both  structures.  What  is  it?  

What  is  the  rmsd  value  of  the  superimposition?  Which  was  the  better  approach  in  this  case?  

Is  the  ligand  of  β1-­‐  (5FW)  binding  at  the  same  cavity  as  the  ligand  of  A2A  (ZMA)?  

 

   

Exercise  4:  Build  an  α-­‐helix  

Build  the  following  sequence  “ERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFC”  as  a  α-­‐helix  using  the  following  command  

fab  ERARSTLQKEVHAAKSLAIIVGLFALCWLPLHIINCFTFFC,  TM6,  ss=1    Display  TM6  as  a  cartoon  

Open  3PWH  

Display  only  helix  6  of  3PWH  from  E219  to  C259  

Superimpose  E1-­‐C41  of  the  build  helix  “TM6”  to  E219-­‐C259  of  3PWH  

We  can  superimpose  only  certain  amino  acids  of  both  molecules  (only  accessible  via  the  command  line):  

Type  “super  tm6  and  resi  1-­‐41,  3PWH  and  resi  219-­‐259”    

Do  they  superimpose  properly?  What  is  the  rmsd?  

Superimpose  E1-­‐L23  of  the  build  helix  “TM6”  to  E219-­‐L241  of  3PWH  

Do  they  superimpose  properly?  What  is  the  rmsd?  

Are  both  helices  identical?