Notes while viewing the crystal structure of an enzyme

• This is a structure measured by X-ray crystallography
• Conformational searching would be such a vast problem for a protein structure, that modelling starting from an arbitrary conformation is not practicable
• While details of a structure may be modelled to fit NMR NOE measurements, most published structures are from crystallographic measurements
• The grey wiggly line in the initial view is the backbone of the enzyme thymidylate synthase
• This enzyme is involved in DNA replication
• In a backbone view, the polypeptide chain is represented by (imaginary) bonds joining the alpha carbon atoms of adjacent amino acids
• The multicoloured ball-and-stick molecules are deoxyuridine monophosphate (known as DUMP), which is involved in the reaction on this enzyme
• The colours are BWT's element colours:  grey for C, blue for N, red for O, violet for P, which are a slight improvement on the standard 'CPK' colours for this particular exercise.  They are set up in an .spt file
• Hydrogen positions were not measured in this crystal structure, so they are not shown
• Chime can calculate probable H positions, if one wishes to look at the detailed environment of a particular atom, but, in general, the picture is cluttered enough without them.  We shall not look at hydrogens in this exercise
• The green ball-and-stick molecules are the anti-cancer drug CB3717, which is blocking a cofactor position
• The .spt file leaves the enzyme part of the structure selected, so that you can change its representation without changing those of  DUMP or CB3717
• The aims of this exercise are to introduce you to commonly used representations in Chime/Rasmol, and to explore how DUMP fits into the enzyme structure

Instructions

• Display Wireframe
• You now see all the measured atoms of the enzyme, in their element colours
• Color Chain
• This shows that the enzyme consists of two chains, chain A coloured blue and chain B coloured green
• There is a molecule of DUMP and a molecule of CB3717 enclosed in each chain
• We will look at the B (green) chain
• Zoom in, using shift left drag downwards, until the DUMP molecule occupies half of the screen height
• Move the DUMP molecule to the centre of the screen, using control right drag
• You can see which amino acid residue is which:  first simplify the picture by going back to the backbone view, using

Display Backbone
• Color Amino Acid
• This is a traditional colour scheme in which the alpha carbon atoms represented by the backbone view are coloured according to which amino acid they belong to, e.g. cysteine and methionine, the sulfur-containing amino acids, are coloured yellow
• Left click on one of the yellow atoms:  you will see the abbreviation CYS or MET and the residue's position in the chain reported at the bottom of the screen
• Cysteine 146 is covalently bonded to the DUMP molecule
• Find cysteine 146
• Using left drag, gently turn the molecule so that you can see this cysteine and the part of the DUMP molecule nearest to it, through a gap in the structure, with nothing in between them
• Now display the whole of the cysteine (except for the hydrogens) and colour the atoms according to element colours:

Display Wireframe
Color CPK
• You should now see the yellow sulfur of the cysteine
• Switch to distance measuring mode, by

Select Mouse Click Action Distance
• Use left click on the sulfur, then on the nearest atom of DUMP, to measure the distance between them, which is reported at the bottom of the screen.  You should find a distance of 1.824 Angstroms.  Compare this with the distance between the sulfur and the carbon of cysteine to which it is bonded.  Clearly, both contacts are C-S single bonds
• Create a molecular surface on the enzyme, by

Select Display List Create Molecular Surface
• This takes a few seconds
• So that you can see it more easily, colour it by

Select Display List Color Cyan
• Zoom out using shift left drag upwards (slowly), until you can see most of the enzyme, with part of the (green) CB3717 hanging out of a hole in the structure.  Turn the model (left drag) until you are looking straight down the hole.  You can glimpse the coloured DUMP molecule behind the green CB3717.  You may need to centre the hole on the screen, using control right drag.  Zoom in again so that you can see down the hole well
• You can now cut away the front of the surface, to reveal the DUMP and CB3717 molecules nestling inside
• Options Slab Mode
• Slab mode usually comes up with most of the molecule cut away, so come back to the intact surface using

control left drag downwards
• Now slowly take slices away from the front of the model, by gently doing control left drag upwards
• You should cut away the green CB3717 molecule piece by piece until it has gone, but the intact DUMP is left behind it
• You can now see how DUMP fits into the enzyme
• If you have time to wait for at least a few minutes of calculation, during which your PC will be locked up, you can colour the whole surface of the enzyme with its electrostatic potential
• Select Display List Color Electrostatic Potential Color Red-White-Blue  (do not use the Rasmol Potential Color Scheme, as in the LUMO exercise earlier in this course:  it is so gaudy that it is difficult to see the guest molecules against it)
• In the red-white-blue color scheme, blue is the most positive potential (the opposite way round to Rasmol)
• You can see that the (negatively charged) 1-coordinate oxide ligands of the phosphate group in DUMP are held in a hole feeling the most positive potential
• Now change the selected part of the model, and display the DUMP molecule in space-filling mode:
• Select Residue UMP
• Display Spacefill Van der Waals Radii
• You can now see that there is an almost exact fit of the enzyme surface to the shape of the molecule

Alpha helices, beta sheets, etc.

• Return to the original view by pressing your browser's reload button
• Display Cartoons
• Color Structure
• Zoom in to look at the B chain region, as before
• Alpha helices and beta sheets are displayed as ribbons
• Turns are displayed as ropes
• Alpha helices are coloured magenta-red
• Beta sheets are coloured yellow
• Turns are coloured blue
• Switch off the hetero groups by Options Display Heteroatom Groups
• Colour code according to the position in the protein chain, by Color Group
• The chain is now coloured in rainbow colours, from red at the carbon terminus (i.e. COOH group) to blue at the nitrogen terminus (i.e. NH₂ group)
• See if you can find the red terminus, zooming and turning as necessary
• Turn the molecule so that this terminus sticks out into clear space
• You can colour the cartoon representation according to amino acid type, as you did for the backbone representation:
• Color Amino Acid
• Left click on the terminal amino acid to identify it:  it should be ILE 264
• Check that this is a CO2 group by Display Wireframe and Color CPK and clicking on each of the terminal atoms
• One of the oxygens is labelled T for terminal