The webpage requires you to upload a file containing the protein structure you wish to visualise. Such files are stored in the Protein Databank (PDB) and are called PDB files. The PDB and PDB files are discussed in the Teaching Portal. You can (1) upload a PDB file from your computer, (2) upload an EZM file containing a previous EzMol session or (3) use a unique PDB ID code to retrieve a PDB file from the databank.

Haemoglobin is the protein that transports oxygen around the bodies of humans (and most other vertebrates). Sickle cell anaemia is an inherited disease resulting from a mutation in the haemoglobin protein. It causes haemoglobins to clump into fibrils which distort the shape of red blood cells, making them “sickle-shaped”.

For our step-by-step example we will use PDB entry 2HBS, which is the structure of a two haemoglobin proteins clumped together as a fibril.

Go to the EzMol start page, enter 2HBS as the PDB ID code and press "Submit". The oxygenated haemoglobin model will be loaded.

Moving the protein

On the left of the screen is a white square containing the protein (Figure 2). As you explore EzMol, this square will remain present and allow you to visualise the protein. You can rotate the protein by left-clicking and dragging it within the visualiser, or by using the options in square (1) (right panel). You can zoom in and out using the right click (and dragging the protein) or square (2) (middle panel) and you can drag the protein by clicking with the wheel of the mouse or square (3) (left panel).

You can turn on and off a rocking or rolling motion by clicking the corresponding buttons. The ‘Enlarge’ and ‘Shrink’ buttons change the size of the visualiser. You can always go back to the default view by clicking ‘Reset camera’.

EzMol provides you with some information about the structure you are viewing, including the name of the structure, its PDB ID and the date it was deposited into the PDB.

The tab ‘More details’ provides you with extra information, including the number of chains and residues, the organism the molecule is from, the experimental techniques used to solve the structure, the resolution, the molecular weight of the molecule and the people who published the structure.

To move to the other steps in the EzMol workflow, use the top navigation bar shown in Figure 3 below.

In the top navigation bar, click on ‘Step 2 - Chain style’. You can change the style of each chain separately.

The cartoon display only shows the backbone of the protein (and not the side chain atoms) according to secondary structure.

The stick display, on the other hand, shows the bonds between all atoms other than hydrogen in the molecule (including side chain atoms). Check ‘Colour stick heteroatoms by element’ to show different atoms with different colours. This allows easy identification of the atoms making up biological molecules.

Chains can be specifically hidden, allowing the visualisation of only part of the molecule. Each chain can also be displayed as a surface, which shows the space occupied by the molecule.

Use the display table to apply surfaces to A and B, hide all chains except C and D, display the heteroatoms as sticks, and ‘Colour stick heteroatoms by element’ (as in Figure 4).

This will display a single haemoglobin protein with one set of the alpha and beta subunits as a cartoon, and the second set of alpha and beta subunits with a surface. The iron-containing heme groups bound to the alpha subunits are displayed as sticks.

You can change the background colour. When saving and printing a view of a protein (cf. Step 8), it is generally advised to use a white background.

You can colour the cartoon representation, the sticks and the surface of each chain. This is a particularly useful way of conveying information about the different polypeptide chains constituting a protein, as shown in Figure 7 below.

Select a red colour for the surfaces of chains A and B, and select a light blue for the cartoons of chains C and D.

Colouring individual residues

You can select a colour from the drop-down menu and then select the residues you wish to display in that colour. You can also choose to hide residues by selecting the eraser and then selecting the appropriate residues.

Secondary structure elements

By clicking on ‘Apply by secondary structure type’, you can choose to colour specifically all residues in α-helices, β-strands or coils, as shown in Figure 8. Teaching resources about protein secondary structure are available in the Teaching Portal.

The mutated residues are Valine 6 in chains B and D. Highlight the valine 6 residue in Chain D in yellow.

You can select a colour and then select specific amino acid residues to display their side chains as sticks (of the colour you have selected). You can select the eraser and then select specific amino acid residues to hide their side chains.

This is useful as the relative position of two side chains (or a side chain and the backbone, for instance) is sometimes functionally relevant, e.g. to form a salt bridge between residues.

Display the valine 6 residue in Chain D in yellow.

You can select a colour and then select specific residues whose surface you would like to highlight. If the protein was already shown as a surface, then the patches corresponding to the residues you have selected will simply adopt the chosen colour. However, if the protein was originally displayed as a ribbon or sticks (which you can change in ‘Step 2 - Chain style’), then a transparent surface will be generated for the entire protein but only the selected residues will be coloured.

Click on ‘Apply by scheme’. You can colour the surface according to one of three characteristics: the temperature factor (B-factor), hydrophobiity and charge. The B-factor is a measure of how flexible an atom is in the structure. A high B-factor indicates that the atom moves a lot, whereas a low B-factor shows that it is fixed in space.

You can also set the colour scheme. If one chain was hidden, it will appear when you select one of the three schemes, you can hide it again by going back to ‘Step 2 - Chain style’ and unclicking ‘Display surface’ for that chain.

Highlight the surface of chain B in yellow.

This tab allows you to label the amino acid residues in the protein. There are several options for the type of label you want to display and you can change the colour of the text (foreground) and the label background. You can choose to remove the label background by making it transparent (last square in the colour menu).

To select which residues to label, click ‘Apply to individual residues’. Labelling important residues is very useful to produce informative figures, which you can save as explained below (‘Step 8 - Render and download’).

Label the Valine 6 residues in chains B and D.

At any point during your work, you can click on ‘Step 8 - Render and download’ (in the top navigation bar) and then ‘Click here to save the image’. This will save a PNG file of the view currently displayed in the visualiser.

You can also click on ‘Save your work log’ to download an EZM file containing the changes you have made during your session (e.g. changing the display style or the colour of the protein). You can then load that file later as described in Step 1.