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Structural templates

2010-05-11T10:54:27Z

James D Watson: New page: ==Motifs In Proteins== <applet load='1aay' size='350' frame='true' align='right' caption='Zinc fingers'/> The term "motif" when used in structural biology tends to refer to one of two cas...

==Motifs In Proteins==
<applet load='1aay' size='350' frame='true' align='right' caption='Zinc fingers'/>
The term "motif" when used in structural biology tends to refer to one of two cases:
<OL>
<LI>A particular amino-acid sequence that characterises a biochemical function
<LI>A set of secondary structure elements that defines a functional or structural role
</OL>

There are a great number of protein sequence motifs identified, many of which have well defined structural or functional roles. One such example of this is the so-called '''[http://www.ebi.ac.uk/interpro/IEntry?ac=IPR007087 zinc finger motif]''' which is readily identified from the following consensus sequence pattern (where "X" represents ''any'' amino acid): 

'''Cys''' - X(2-4) - '''Cys''' - X(3) - Phe - X(5) - Leu - X(2) - '''His''' - X(3) - '''His''' 
 

The example structure shown to <scene name='User:James_D_Watson/Structural_Templates/Zinc_finger_highlight/1'>illustrate the motif</scene> is that of Zif268 protein-DNA complex from Mus musculus (PDB entry 1AAY). In this example (a C2H2 class zinc finger) the conserved <scene name='User:James_D_Watson/Structural_Templates/Zinc_finger_cysteine/1'>cysteine</scene> and <scene name='User:James_D_Watson/Structural_Templates/Zinc_finger_histidine/2'>histidine</scene> residues form ligands to a <scene name='User:James_D_Watson/Structural_Templates/Zinc_finger_zn/1'>zinc ion</scene> whose coordination is essential to stabilise the tertiary fold of the protein. The fold is important because it helps orientate the <scene name='User:James_D_Watson/Structural_Templates/Zinc_finger_recognition/1'>recogniton helices</scene> to bind to the <scene name='User:James_D_Watson/Structural_Templates/Zinc_finger_major_groove/1'>major groove of the DNA</scene>.
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However, there are also a number of repeated patterns and functional motifs revealed when the protein structure is examined. This page aims to introduce some of the main types of motif illustrating them on protein structures from the PDB.

==Secondary structure elements==

Proteins are formed from linear chains of amino acids joined together by peptide bonds. These chains then fold up to form the three dimensional shape. However, the relative rigidity of the peptide bond combined with the presence of amino acid sidechains, means that not all conformations are acceptable and there are many cases where the various atoms in the chain start to collide with one another. Two of the most stable (and therefore most commonly observed) conformations are the '''α-helix''' and the '''β-pleated sheet'''. These along with a number of small turns in the chain and random coil (folds that do not fit into a classification) are known as secondary structures. 

===α-helices===
<applet load='5p21' size='350' frame='true' align='right' caption='Secondary structure - Alpha' scene='User:James_D_Watson/Structural_Templates/Secondary_structure_start/1'/>
In the Jmol viewer to the right PDB entry 5p21 has been coloured by secondary structure (α-helices are coloured magenta and β-strands are coloured yellow). The <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_alphahelix/1'>α-helix</scene> is formed when the amino acid backbone forms a right handed spiral with 3.6 amino acids per turn. The <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_alpha_sc/1'>sidechains point outward</scene>, away from the centre of the helix, where they can interact with solvent, other protein, small molecules or macromolecules. The structure is stabilised by regular hydrogen bonds that form between the backbone carbonyl oxygens and amide hydrogens. The bonding pattern for the α-helix is characterised by the <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_alpha_hbon/1'>carbonyl group of residue i interacting with the amide group of residue i+4</scene>, this is known as an (i, i+4) interaction. The alpha-helix can take other less common forms including π-helices, 310-helices and their left handed forms (see table 1 for the helix parameters). 
 
Table 1: 
<table border=1>
<tr>
<th>Type of helix</th>
<th>Bonding pattern</th>
<th>Residues per turn</th>
<th>Rise per residue</th>
<th>Radius of helix</th>
</tr>
<tr>
<td>310-helix</td>
<td>(i, i+3)</td>
<td>3.0</td>
<td>2.0</td>
<td>1.9</td>
</tr>
<tr>
<td>α-helix</td>
<td>(i, i+4)</td>
<td>3.6</td>
<td>1.5</td>
<td>2.3</td>
</tr>
<tr>
<td>π-helix</td>
<td>(i, i+5)</td>
<td>4.3</td>
<td>1.1</td>
<td>2.8</td>
</tr>
</table>
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===β-sheets===
<applet load='5p21' size='350' frame='true' align='right' caption='Secondary structure - Alpha' scene='User:James_D_Watson/Structural_Templates/Secondary_structure_start/2'/>
In the Jmol viewer to the right PDB entry 5p21 has been coloured by secondary structure (α-helices are coloured magenta and β-strands are coloured yellow). A single <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_beta/1'>beta-strand</scene> can technically be described as a flat helix with 2 residues per turn although this may not be initially obvious. 
When two or more beta strands lie next to each other, forming hydrogen bonds between them, this is what is termed a <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_betasheet/1'>β-sheet</scene>. As the backbones need to come close together to interact and form a sheet, the <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_beta_sc/1'>sidechains are oriented away from the plane of the sheet</scene>. As the polypeptide chain is synthesised from the amino terminus to the carboxyl terminus it has a directionality (represented in the cartoon format as an arrowhead on each beta strand). β-sheets therefore occur in two varieties:<OL>
<LI><scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_beta_ap/1'>Anti-parallel</scene> - here the beta strands aligned next to each other run in opposite directions. As the interacting carbonyls and amides align well, the hydrogen bonds appear to be straight.
<LI><scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_beta_para/1'>Parallel</scene> - here the interacting strands run alongside each other and point in the same direction. In this conformation the carbonyl oxygen and the amides tend to be more staggered than in an anti-parallel sheet, therefore the hydrogen bonds tend to be angled. Also note the rare (i, i+2) bond within the lower strand.
</OL>
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===Turns and loops===
<applet load='5p21' size='350' frame='true' align='left' caption='Secondary structure - Alpha' scene='User:James_D_Watson/Structural_Templates/Secondary_structure_start/3'/>
There are a number of small hydrogen bonded motifs and patterns which are observed regularly. These are described below:<UL>
<LI>'''<scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_betaturn/1'>Beta Turns</scene>''' - originally defined by the one hydrogen bond common to all (an i, i+3 hydrogen bond) but some modern descriptions do not require a hydrogen bond.
<LI>'''Beta Bulge Loops''' - often associated with beta sheets and result from an additional residue being found in one strand. This interrupts the regular hydrogen bonding and causes a distinctive bulge.
<LI>'''Alpha turns''' - the simplest of all motifs and is characterised by one (i, i+4) hydrogen bond. It is found as part of the hydrogen bonding network of alpha helices as well as occurring on its own.
<LI>'''<scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_paperclip/1'>Paperclip/Schellman Motifs</scene>''' - a common motif found at the C-termini of alpha helices which is essentially a reverse turn that breaks the alpha helix out of its cycle. It is characterised by the presence of a left handed residue and two hydrogen bonds: an i, i+3 bond and an i, i+5 bond.
<LI>'''Gamma Turns''' - these rarer type of turns are characterised by an (i, i+2) hydrogen bond, which is rather weak because of the bent geometry involved.
</UL>
 
 
 
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<applet load='1lmb' size='300' frame='true' align='right' caption='Lambda repressor' scene='User:James_D_Watson/Structural_Templates/Helix_t_helix/1'/>
These secondary structure motifs can be combined to form functional motifs, the most well known of which is the helix-turn-helix motif found in a number of DNA-binding proteins. The computational identification of these motifs is straightforward but made complicated by the fact that not all helix-turn-helix motifs bind DNA. The problem faced here is therefore one involving the distinguishing between true and false positives. The structure to the right is that of lambda repressor bound to DNA. The helix-turn-helix motif is readily identified in green.

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==Nests==
Smaller than loops and turns are some recently discovered motifs known as "nests". These are mainchain conformations where 3 successive amide groups form a positively charged concavity capable of binding one or more negatively charged atoms (Figure 1). They are characterised by alternating enantiomeric mainchain dihedral angles from the alpha and gamma regions of the Ramachandran plot, and can be of RL (right handed - left handed) or LR type. They are most commonly found as part of previously described hydrogen bonded structural motifs but are also found at functional sites. 
 
[[Image:Nest 1np4.gif]]

These basic units can be combined in succession to form more complex motifs and come in two categories:<OL>
<LI>Compound nests - occur where the nests overlap so that the residues alternate between R and L forms (e.g. RL, RLR, RLRL)
<LI>Tandem nests - where two nests sit side by side (e.g. RLLR, LRRL, RLLRRL)
</OL>

In compound nests the result is a long chain with all the overlapping nests facing a similar direction. This basically forms a much wider nest that is capable of binding a larger anionic group of atoms such as the phosphate ion, and are usually functionally important motifs. Tandem nests are not as common and, due to the greater change in the direction that adjacent nests face, only seem to perform functional roles when found in conjunction with one or more compound nests.

<applet load='1lmb' size='300' frame='true' align='left' caption='Nest in PDB entry 5p21' scene='User:James_D_Watson/Structural_Templates/Secondary_structure_start/4'/>

One of the most well known functional compound nests is found in the phosphate-binding loop of Ras protein (PDB entry 5p21). The <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_ploop/1'>P-loop</scene> is a well described ATP- or GTP-binding loop present in a large superfamily of important proteins which includes G-proteins and kinases. The main feature of the P-loop is a long compound LRLR nest that <scene name='User:James_D_Watson/Structural_Templates/Secondary_structure_ploop_nest/1'>forms a binding site for the β-phosphate of ATP or GTP</scene>. However, this is an example of a motif where the ligand also binds to the free main chain NH groups at the N-terminus of an alpha helix. On closer inspection it becomes evident that this interaction is in addition to the compound nest and does not interfere with it. Therefore the P-loop is actually more accurately described as a compound LRLR nest and an adjacent helical N-terminus that collectively bind to the α- and β-phosphates of the GDP substrate. The P-loop, which is retained throughout the superfamily, has a highly conserved GxxxxGKS/T consensus sequence (where the xxGK section forms the LRLR compound nest).
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==Templates and Active Sites==

Moving away from secondary structure elements, loop and nests, another type of structural motif is that of enzyme active sites. These structural motifs are usually more difficult to detect as they can be discontinuous, often involving elements widely spaced along the sequence. One such example is that of the "catalytic triad" of the serine proteases.

Serine proteases are found in a number of organisms but common to their function is the hydrolysis of peptide bonds. These enzymes catalyse the reaction using a highly reactive serine residue to attack the carbonyl group of the backbone to be hydrolysed. The chemistry of this reaction and the regeneration of the active site, requires the presence of the Ser-His-Asp catalytic triad. In chymotrypsin (PDB entry [[1ab9]]) these residues are (Ser-195, His-57 and Asp-102) whereas in the bacterial subtilisin (PDB entry [[1st2]]) the site is formed by (Ser-221, His-64 and Asp-32). These two proteins are evolutionary unrelated and this is the classic example of convergent evolution to solve the problem of peptide bond hydrolysis.

The detection of these types of motif is almost impossible by looking at the amino acid sequence: there is no evolutionary relationship to detect, the residues are ordered differently in the sequence, and the spacing between the residues also varies. These motifs can be detected relativeley easily using structural comparison, particularly template-based motif detection algorithms. The subtilisin and chymotrypsin structures are shown side by side - note that the global folds of these two proteins are very different so the site could not have been detected using such methods. Click to see the catalytic triad in <scene name='User:James_D_Watson/Structural_Templates/Subtilisin_startpoint_catalyti/1' target='subtilisin'>subtilisin</scene> and <scene name='User:James_D_Watson/Structural_Templates/Chymotrypsin_start_triad/1' target='chymotrypsin'>chymotrypsin</scene> respectively.
<applet load='1st2' size='300' frame='true' align='left' caption='Subtilisin 1st2' scene='User:James_D_Watson/Structural_Templates/Subtilisin_startpoint/1' name='subtilisin' />
<applet load='1st2' size='300' frame='true' align='right' caption='Chymotrypsin 1ab9' scene='User:James_D_Watson/Structural_Templates/Chymotrypsin_start/1' name='chymotrypsin' />

2009-02-10T00:41:20Z

James D Watson:

==Introduction to Jmol==
Jmol is a commonly used molecular graphics viewer. The aim of this tutorial is to provide you with an introduction to some of the common manipulations to help you understand protein structure data. 
 
There are three main ways for you to interact with protein structures using Jmol:<UL>
<LI>the mouse
<LI>the menu
<LI>the console
</UL>
 

==The Mouse==
<applet load='1aay' size='350' frame='true' align='right' caption='Practice movements'/>
 
The three elementary ways to interact with the molecule in the viewer are:<OL>
<LI>Rotation
<LI>Translation
<LI>Zooming
</OL>
 
The most basic interaction with a molecule involves rotating it around in the viewer. To freely rotate the molecule yourself, left-click and drag on it. Drag the cursor up and down for x-axis rotation, left-right for y-axis rotation. 
 
Occasionally it is necessary to translocate the protein along an axis. In order to do so yourself, hold down shift, then double-click and drag on the structure. You will see the molecule follow your mouse movements (up, down, left and right). 
 
It is common to zoom in on particular aspects of a molecule. To zoom the molecule yourself, shift-click and drag. Drag the cursor down to zoom in, drag it up to zoom out. 
 
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==The Menu==

In this section we will be looking at the structure of the human deoxy-haemoglobin protein (PDB entry 4HHB). The display shown here clearly shows the folding patterns of the polypeptide chains, and the arrangement of the chains with one another. However, it does not give you an accurate picture of the shape of the molecule, nor the significance of the bound ligand. In this tutorial we will use the menu options to change the display in order to highlight these different aspects. Throughout this section, we will represent selections using the Jmol menu in '''''this style'''''. 
 
 
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=== Selecting and rendering ===

<applet load='4hhb' size='400' frame='true' align='right' caption='Haemoglobin molecule' scene='User:James_D_Watson/Using_Jmol/Haemoglobin_start/1'/>
In order to access the Jmol menu click on the word "Jmol" in the lower right corner of the structure display area. The main panel of the Jmol menu will appear. (To close the menu without using it, click anywhere else in the structure window). 
 
It is vitally important to understand that all actions are performed on what was last selected. If the display does not change as you expect it to, doubly check what you have selected, make the selection again and redisplay. To select something via the main panel of the Jmol menu, move the cursor over the word '''''Select'''''. This will bring up a selection of sub-menus, click on '''''All'''''. The whole menu will disappear, and Jmol will perform the task of selecting all the atoms in the structure, please note that the display will not change yet. 
 
 
In order to change the display you need to "render" your selection. To render, access the Jmol main menu again and place the cursor over '''''Style''''' to see its sub-menu. Move the cursor to '''''Atoms''''' then click on '''''100% van der Waals'''''. The menu will disappear and the display will change so that each atom is represented as a solid sphere (commonly called "spacefill"). 
 
Try this for yourself and if you want to check that your menu selections worked correctly you can click on the "Check Section" links. These will change the Jmol image to show what the menu selections should have resulted in. 
 
From now on we will represent successive menu selections in the following way: 
'''''Select | All''''' 
'''''Main | Style | Atoms | 100% van der Waals''''' 
 
 
This will repeat the steps taken above. <scene name='User:James_D_Watson/Using_Jmol/Haemoglobin_checkpoint1/1'>"Check Section"</scene>. 
 
If you wished to undo the above selection then make the following menu choices: 
'''''Select | All''''' 
'''''Main | Style | Atoms | Off''''' 
 

=== Selecting and rendering ===

Displaying the entire protein as spacefill spheres gives a better impression of the actual shape of the protein. In this case the four subunits are clearly packed together with little space between them. It is also evident that there are a number of small molecules bound to the protein surface. These are often solvent molecules (water, deuterium, etc) or artefacts of the crystallisation process (glycerol, etc), but they can also represent substrate mimics, inhibitors and cofactors. In the example shown there are a number of water molecules which is confusing the identification of any ligands. To remove these atoms: 
'''''Select | Hetero | All Water''''' 
'''''Main | Style | Atoms | Off''''' 
 
This leaves the remaining bound small molecules. Explore the structure some more to confirm the identity of these tightly bound molecules as the haem groups. <scene name='User:James_D_Watson/Using_Jmol/Haemoglobin_checkpoint2/1'>"Check Section"</scene>
 
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What if you only want to see the protein, without the pigments?

Select | Hetero | Ligand
Render | Bonds | Off

The pigments (which are ligands of this protein) were displayed as bonds only, so turning off the bonds turned off the display of the pigments altogether.

Now let's redisplay the pigment, this time as a "ball and stick" model, and also with the atoms colored by element.

Select | Hetero | Ligand
Render | Scheme | Ball and Stick
Color | Atoms | Scheme | Element (CPK)

Zoom in using the mouse to get a better look at the atoms of the pigments.

Showing specific amino acids:

Sometimes, you may want to look at a certain amino acid or a type of amino acid in a molecule. For example, some proteins are stabilized by disulfide bonds, which are covalent bonds formed between two cysteine side chains. Three disulfide briges are present in the protein insulin. First, we'll look at the overall structure of insulin.

As you see, insulin is quite a small protein. To the see disulfide bonds, use the Jmol menu in the sequence below:

Select | Protein | All
Render | Atoms | Off
(Don't worry, it's supposed to disappear!)
Render | Structures | Cartoon
Select | Protein | By Residue Name | CYS
Render | Bonds | 0.3 Å
Color | Disulfide Bonds | Yellow

Note that that the cartoon and stick renderings would have been hidden by the spacefilled atoms if you had neglected to turn them off. This is because the current rendering choice is not automatically turned off when another choice is selected; each rendering style is controlled individually, so multiple display styles of the same atoms are possible.

You can also change the display of disulfide bonds under Render | Disulfide Bonds, but note that they must be part of the currently selected set of atoms to be affected by the change.

Inverting selection

What if you want to change the display of everything except the cystines?

Select | Protein | By Residue Name | CYS
Select | Invert Selection
Color | Cartoon | Orange

The Jmol Console
Jmol can also be controlled using a command language. Knowing even a few simple commands can useful. To access the Console, use the Jmol menu:

Console... | Open

A small window, the Jmol Console, will appear at the upper left corner of your screen. A cursor will be blinking in the lower part of the window. This is where you can enter commands.

Type the following command:
select all
and click the "Execute" button on the Console. Jmol writes a few things into the upper part of the Console window after executing the command, including the number of atoms selected.

Now type:
color purple
and click the "Execute" button again.

And finally, type:
spacefill 100%
and click "Execute".

The Console allows very powerful control of Jmol with the command language. You can see a listing of Jmol commands at the Jmol interactive scripting documentation page (opens a new window).

Now that you are more familiar with Jmol, click below to move to the section on protein secondary structure.