Introduction to Swiss Pdb Viewer:
Practical 1 within the course 1MB580, "Macromolecular structure and function" (Molecular Biotechnology, Uppsala University 2008)
The structure levels in proteins
The purpose of this computer exercise is to give an introduction to the molecular graphics program Swiss Pdb Viewer and to protein structures and their building blocks. You will learn how to display proteins in different ways, how to select and display different parts of the protein, how to superimpose structures and how to measure distances.
A first look at protein structure-function relationships will be done by looking closer
at the oxygen binding proteins
myoglobin and hemoglobin.
- Work alone or in pairs during the computer practicals.
- Those students that attend and complete the practical, will immediately get their answers checked by one of the assistants present, and if possible also get the practical approved there.
- Those students that do not attend one of the scheduled computer practicals, or for
some other reason don't complete one of these, should e-mail in a full report as soon as possible.
Hemoglobin and myoglobin
All globin proteins are closely related both in sequence and in three-dimensional structure.
function of the myo- and hemoglobins is to bind oxygen. They both consist of globin fold
polypeptide chains with a bound heme group. The heme group is a so-called prosthetic group and is indispensable for the oxygen binding function of the protein. The heme consists
of four pyrrole rings joined by methene bridges with an iron atom in the centre.
Myoglobin is the red pigment in muscles. The role of myoglobin is to take up oxygen from the blood and
function as a reservoir of oxygen in the muscle cell and other tissues. The skeletal muscle of diving
mammals is particularly rich in myoglobin, which serves as a store of oxygen during a dive. The first
protein structure ever to be solved was that of sperm whale myoglobin. This was done in 1957 in a now
classical work by John Kendrew and his coworkers in Cambridge, UK. At this time, myoglobin from the
skeletal muscle of sperm whale was selected for X-ray crystallographic studies because it could be
isolated in large amounts and forms excellent crystals. Nowadays most structural studies are performed on recombinant protein.
The PDB and pdb files
Coordinates of biological macromolecules are usually written in as simple text format, the 'pdb-file' format. Protein coordinates are stored in databases with links to other information such as sequences, publications etc.
The coordinates of most of the structures solved are collected in a database called the Protein Data Bank: PDB @ RCSB. You will learn more about the PDB in practical 3.
All the files that you need can be downloaded here.
- Open UserDisk (H) and create a folder where you can store your files.
- Right-click and select "Save link as..." to save the files locally.
- Save the files in your own directory and please keep the names of the files.
Basics of Swiss-PdbViewer
A user's guide to this program is avaiable at http://www.expasy.ch/spdbv/text/ctrlpanl.htm.
In the graphics window, the protein myoglobin should appear.
- Locate the Swiss-PdbViewer program on this computer. Start the program. After a short while an
"open PDB file" dialogue should appear. Select the file 1a6n.pdb.
You can also open a pdb file using "CTRL + O" (i.e., by pressing the CTRL button at the same time as the letter "O"), or "Open PDB file" in the File menu.
You will use two different windows. The protein you just opened is displayed in the
On this page; you can find a manual for
what can be done using the different buttons in the graphics window. Now try these:
The control Panel is a second very useful window. If it didn't appear when you opened your pdb file, you can find it under the Wind-menu.
- Rotate and translate the molecule.
- Zoom in and out.
- Center on different parts of the molecule
This page of the Swiss-PDBViewer manual tells you everything about the control panel.
You will see several columns. You should see a list of all the amino acid residues in
Two important things: when an amino acid is couloured red it is selected, when it is black it is not. If you press return on your keyboard, only the selected amino acids will be shown.
Selecting more than one residue - try these different variants.
- Try clicking on one of the amino acids and then press return.
- Now select amino acids 1-10 and press return.
The slab function.
- To select all residues in the pdb file: go to the select menu and choose all or use "CTRL+A" (Note that sometimes there are multiple polypeptide chains within one pdb file; named A, B, C...).
- Selecting neighboring residues (1): in the control panel, drag the cursor over the residues you want to select.
- Selecting neighboring residues (2): in the control panel, select the first one, press <Shift> and select the last one.
- Selecting non-neighboring residues: In the control panel, press <Ctrl> and select residues with the cursor + mouse click.
- To mark all the selected amino acids in the "Show" or "Side" column, click on the word "Show" or on the word "Side".
- To unmark all the amino acids selected in the "Show" or "Side" column, press <Ctrl> and <Shift> at the same time and
click on the word "Show" or on the word "Side".
- There are several other handy ways to select residues, e.g., based on secondary structure, type of amino acid residue, etc., which will be described when needed. Some of these are found in the Select menu.
When you center on an atom in the middle of the protein it is difficult to see what you want because the center is in the middle of a forest of atoms. When you use the slab function, you only display a slice of the molecule, i.e. the closer part and the further part are invisible. This will be a very useful function when you examine different proteins during the tutorials.
- Try selecting Display => Slab. You should only see a z-slice of your molecule.
- Try pressing the shift button and moving the cursor up and down. Now different slices of your molecule are visible.
- If you loose your molecule, turn off the slab and re-center. If needed, you can change slab settings under Prefs => Display.
There are four different levels of structures: primary, secondary, tertiary and quaternary structure.
The primary structure is the sequence of amino acids in the protein or peptide (one-dimensional). As you know by now, each amino acid has both a three- and a one-letter abbreviation. SwissPdbViewer uses the three-letter abbreviations.
Q1. Write down the one-letter abbreviations for amino acids 15, 35, 37, 53, 118, 130, 150, 28, 132 and 136 in the a6n structure. Together they form a word.
The secondary structure is a regular structural unit formed by sequence regions in a protein (from a sequence perspective: two-dimensional).
Basically, these are of two types: α-helices and β-sheets. There are also a number of less common structural motifs that
sometimes are regarded as secondary structures (e.g., β turns, left-handed helices).
Now colour the protein based on atom type (CPK).
Now you will explore some properties of α-helices.
In the Display menu, you can select or de-select "Show Sidechains even if Backbone is hidden". When selected, you can display sidechains without the backbone visible. When it is not selected, you can display either the backbone atoms (only) or the backbone+sidechain.
The atoms and bonds are now coloured so that; greyish = C, red = O and blue = N. Hydrogens are missing (should be coloured cyan).
Hydrogens can be added.
- Select all atoms. In the "Color" menu, select "CPK".
Back to the Contol Panel. In some cases there is a letter to the left of your amino acids, it can be either an s or an h.
- Select "Add Hydrogens" in the "Build" menu (ignore and close the error dialogue), then select "Show hydrogens" from the "Display" menu.
Q2. Click on an "h" and press return. What happens and what do these letters stand for? Note that even if you only click on one h or s, all the amino acids close to this one with the same letter in front gets selected.
Q3. How many alpha-helices and how many beta strands can you find in myoglobin?
In this kind of protein fold, the helices are often named by capital letters starting from helix A in the N-terminal.
Look at the helix with and without the sidechains visible (control panel). Helices have a polarity and the direction, N- to C-terminal, is maybe not obvious. Since there is a polarity, the ends of helices are very often located at the surface of protein molecules.
- Select and show Helix E only. Center somewhere in the middle of the helix.
Q4. Try to find out a way/pattern to determine the direction (N->C) of an α-helix and make a simple drawing that indicates this pattern?
Q5. Try to explain why the ends of α-helices can be polar and draw a helix to show the partial charges at the N- and C-terminal ends, respectively (hint: look at the
figure of a peptide bond).
Helices as well as beta-sheets are held
together by a network of hydrogen bonds. Possible H-bonds can be computed based on distances and angles between potential donors and acceptors. All these H-bonds are not necessarily present all the time, since proteins can be very dynamic.
Q6. Describe the main-chain hydrogen-bonding pattern in an α-helix in a simple drawing.
- Compute H-bonds in the "Tools" menu and, if not visible after computation, select to show them in the "Display" menu.
Helices can be left-handed or right-handed. To check this, use your hands. Point the thumb towards the C-terminal along the helix-axis, the fingers should then be able to "grab" the helix pointing towards the C-terminal.
Q7. Is this helix left-handed or right-handed?
Tertiary structure is the three-dimensional fold of the protein, or how the secondary structure elements of the protein are packed against each other.
The pdb file you have loaded is a near atomic resolution model of myoglobin with no oxygen bound, deoxymyoglobin.
The heme group and other "hetero-compounds" (atoms that do not form the protein or nucleic acid chain) can be
found at the end of the Control Panel after the last protein residue.
Q8. How many atoms does the heme iron coordinate in the deoxy state?
- To add the heme group to your display, press "Ctrl" and click on "Hem" in the Control Panel (keep the residues of Helix F selected). Press "Enter".
Q9. What residue in the protein coordinates the heme iron?
Ribbon representation of protein structure
- One way to answer this question is to select the hem, go to "Select" menu -> "Neighbors of Selected aa" ->
"Add to view groups that are within"... e.g. 4.0Å
Q10. Describe how the secondary structure elements are packed together. Are the
same helices close together in sequence and in space?
- Select all residues and show the whole molecule.
- Click on the word "ribn" in the Control Panel
- Open the preferences for "ribbon" in the "Prefs" menu and check the box for "Render as Solid Ribbon".
- In the "Display" menu, check "Render in 3D".
- In the Control Panel, click on the small arrow beneath "col" (keep the mouse buttom pressed). In this pull-down menu, select "ribbon". (In this pull-down menu you choose what to colour: backbone only, backbone+sidechain, sidechain only, ribbon, label or surface.)
- In the "Color" menu, choose to colour the ribbons by "Secondary structure" and "Secondary structure succession". (Check the difference).
Myoglobin with oxygen bound - how to superimpose structures
Load the file "1A6M", which is an (near) atomic resolution model of oxymyoglobin (myoglobin with oxygen bound).
Now we will superimpose the two models 1A6N and 1A6M on the heme group. You can switch between the two models (called "layers" in Swiss-PdbViewer) by clicking the file name in the grey field located in the top of the Control Panel. You can also open the dialogue "Layers info" in the "Wind" menu.
- Select only the two hems and use "Fit selected residues" or "Magic fit"in "Fit" menu, "CA atoms only".
- Select the heme of 1A6N, "Select" -> "Neighbors of Selected aa"
-> "Add to view groups that are within"... e.g. 4.0Å.
- Do the same with 1A6M.
- Press <Ctrl> and <tab> at the same time: by this you
can "blink" between the two layers.
When you go between the two layers, you can see the "breathing" of myoglobin, and the approximate size of the structural differences.
To get an accurate measurment of how much a specific atom moves, do the following.
Q11. What distance does the sidechain of His64 move to accomodate oxygen binding? Use the measuring tool from the toolbar to find out.
- Display both 1a6m and 1a6n with the current selection.
- Center on His64. This residue is pointing to the oxygen bound to the heme group in the 1a6m structure.
- To find the residue, you can label it by checking the labl column in the control panel. You can also right-click on the residue name in the control panel which will re-center on the alpha carbon of that residue.
This file contains the oxy structure. Oxygen is coloured red and the rest
of the molecule is coloured yellow.
- Load the file oxymyoglobin.spdbv.
This is a space-filling static model of oxymyoglobin. In such a model
only exposed residues are seen on the surface.
- Select "Render in Solid 3D" in the Display-menu.
Q12. Try to explain in general terms how release and rebinding of oxygen can come about, though a bound oxygen will be buried in the interior of the protein.
Packing of helices.
We are now going to exemplify a mode of helix packing found
in α-helical structures by looking at helices in the myoglobin structure.
In this file, the helices B and G are displayed. The backbone of helix B
is coloured white. Four of the side-chains in this helix are coloured red.
The backbone of helix G is coloured cyan and four of the side-chains are
coloured yellow. Due to the basic geometry of an α-helix, residues separated by
four in the sequence are close together on the helix surface.
- Close the old layers and load the file helixpacking_bg.spdv.
Q13. Describe the helix packing between the helices B
and G. Estimate the angle between the two helices. Why do you think certain angles are preferred in the close packing of two helices?
- If a dotted surface is displayed, un-check "Show Dots surface" in the
"Display" menu to deselect this option.
- If you find it difficult to see the helix packing select "Show Dots surface
and "Render in solid 3D" in the "Display" menu and look at the structure
Quaternary structure: the way several polypeptide chains together can form a
functional complex. Many proteins "work" together in complexes, e.g., hemoglobin with four subunits or the ribosome that is put together by a large number of separate
protein chains and RNA molecules.
The role of hemoglobin is to transport oxygen from the lungs to
the tissues. Oxygen is bound to hemoglobin in the lungs and transported
to the tissues where it is released. Whereas myoglobin is a monomer, hemoglobin
is a tetramer composed of two copies of two different polypeptide chains,
called the alpha- and beta-chains. Human have genes for several other
subunits of which most of them are expressed in fetus. 98% of the hemoglobin
in human red blood cells has the alpha/beta chains and this is called
hemoglobin A. From both the DNA and the protein sequences of the different
globins, including the myoglobin, it has been suggested that all these
globins have diverged from a common ancestor. The gene duplication events
that should have caused this, happened some 600-800 million years ago (myoglobin
vs. hemoglobin) and about 500 million years ago (alpha- and beta-chain).
Oxygen binding to hemoglobin is cooperative, which means that binding
of some oxygen enhances binding of additional oxygen. The binding of the
first oxygen leads to conformational changes that propagate further through
the hemoglobin tetramer to alter oxygen affinity at other sites.
Myoglobin vs. hemoglobin
As the last part of this tutorial, you will compare hemoglobin and myoglobin.
Q14. What can you say about these folds?.
- Close the previous layers.
- Open the file "3hhb.pdb". This file contains the coordinates of deoxy hemoglobin.
- Select and display only the A chain.
- Open "1a6n.pdb" and select all atoms.
- Superimpose the A chain of hemoglobin with myoglobin using "Fit"=>"Magic Fit".
- Display each of the molecules in a ribbon representation (as above) and color by "Secondary structure succession" (color menu).
Q15. Describe in a simple drawing how the globin enteties are packed together and how the heme groups are oriented.
- Hide myoglobin (tick off "visible" in the Control panel).
- Display the entire hemoglobin in ribbon representation
and color by chain (color menu).
Part of this practical was originally written 2003 by: Henrik Hansson; Gunnar Berglund & Evalena Andersson, Uppsala Universitet and edited 2006 by: Mats Sandgren, Uppsala Universitet.
The rest was written 2007-2008 by: Maria Selmer, Uppsala Universitet.