Chapter 8 in Voet & Voet, 4Th Edition

Chapter 8 in Voet & Voet, 4th Edition

3D Structural Alignment of Proteins at the Protein Data Bank

Structure alignment – lite version. In this exercise, you will compare the structures of serine proteases by aligning the structures in 3D space and observing the similarities and differences.

1.Go to the Protein Data Bank ( and search for 1yph (a chymotrypsin structure).

3.Open two more tabs or windows in your browser. Use one tab/window to find the PDB summary page for 1yph and the other to find the PDB summary page for 1ucy. In each case display the PDB file in the browser window so you can explore it later as part of this problem (click on the Display Files dropdown menu and select PDB file).

4.On the left side of the PDB web site, find the Tool box and click on compare structures.

5.In the web browser, you should now see a box labeled, Compare the following two proteins. Enter the pdb id for chymotrypsin (1yph) and chain (C) on the first line and the pdb id for thrombin (1ucy) and chain (H) on the second line.

6.Select JFatCat Flexible from the ---Select Comparison Method--- dropdown menu and click on the compare button. This will take you to a window that includes a 3D alignment of the structures in Jmol. Identify portions of the chain that are not aligned. You can do this by clicking on the the protein backbone in an unaligned area and looking for the amino acid information in the bottom left hand corner of your browser. You should see something like this: [TRP]148:H.Ca/2 #1400 20.862999 -0.06 11.447. Find this residue in your PDB file in one of the browser windows you opened up in step 3 and see if you can discern the meaning of the terms in this expression.

7.Now let’s find two of the members of the catalytic triad (ser—his—asp) in each structure. Note: the serine is actually located in a different chain, so we’ll be looking for the histidine and aspartate members of the triad.

Below the Jmol window in the browser, there is a box where you can enter Jmol Scripts. You just type in the command, then click on the Jmol Script button. Enter “select his57”.
Enter “color red”.
Enter “wireframe 100”.
Enter “select asp102;color yellow;wireframe 100”.
Enter “select protein;backbone off”. Now zoom in and see how closely aligned his57 and asp102 are in the two proteins.
Save the image you have created.

8.To find the third member of the catalytic triad, repeat steps 4-7 using 1yph chain e and 1ucy chain e, looking for ser195. In this case, if you want to show them in different colors, you have a challenge because this comparison is for chain e in both structures. You will need to use a slightly different selection command to differentiate the two chains. In this web page, the two structures are identified as models 1 and 2. So if you want to select serine 195 from the 2nd model, you use the expression “select ser195 and */2”. Use this to create an alignment of the serine 195 side chains from the two models. Save the image you have created.

Structure alignment – digging a little deeper. This exercise is a more in-depth view of the above exercise using 1yph (chymotrypsin) and a second structure that is not as closely related as 1ucy (thrombin). Note: this is one of those open-ended exercises that will be challenging and may not have a clear-cut answer.

1.Go to the Protein Data Bank ( and search for 1yph (a chymotrypsin structure).

2.Click on the 3D similarity tab in the summary page, then scroll down to the table labeled Entity#2: Chains: C,D. Below that is a second table labeled, 1YPH.C (chain 1) vs. representatives of other sequence clusters (chain 2). Go to page 5 of this table and select a structure that is ranked between 61 and 75 (suggested structures: 1lcy, chain A; 2fyq, chain A; 2zu5, chain A; 208m, chain B). You will have the best results for this exercise if you select a structure that includes the terms protease or serine protease in the title. Caveat: If you find you are frustrated during steps 3-8 by not being able to find the catalytic triad in the second structure you select, then switch structures to something that is higher on the list.

3.If you are not finding the correct residues, you may need to consider some alternatives.

This structure may have been modified to remove one of the catalytic residues so that it could be crystallized without digesting itself. For example, the analog of ser195 may have been replaced with ala or gly (see 1lcy).
The numbering may be different. His57 from 1yph may be analogous to his65 from another structure.
The homolog may not actually be a serine protease, but may be a member of the same superfamily.
Additional resources to consider if you run into problems:
PDB Sum ( simply enter your PDB id and look for useful information.
The Catalytic Site Atlas ( Just enter the PDB id for your structure. If it is present in the Catalytic Site Atlas, you can find the active site residues there.

4.Open two more tabs or windows in your browser. Use one tab/window to find the PDB summary page for 1yph and the other to find the PDB summary page for the other pdb id. In each case display the PDB file in the browser window so you can explore it later as part of this problem (click on the Display Files dropdown menu and select PDB file).

5.On the left side of the PDB web site, find the Tool box and click on compare structures.

6.In the web browser, you should now see a box labeled, Compare the following two proteins. Enter the pdb id for chymotrypsin (1yph) and chain (C) on the first line and the pdb id and chain for your second structure on the second line.

7.Select JFatCat Flexible from the ---Select Comparison Method--- dropdown menu and click on the compare button. This will take you to a window that includes a 3D alignment of the structures in Jmol. Identify portions of the chain that are not aligned. You can do this by clicking on the the protein backbone in an unaligned area and looking for the amino acid information in the bottom left hand corner of your browser. You should see something like this: [TRP]148:H.Ca/2 #1400 20.862999 -0.06 11.447. Find this residue in your PDB file in one of the browser windows you opened up in step 3.

8.Now let’s find two of the members of the catalytic triad (ser—his—asp) in each structure. Note: the serine is actually located in a different chain in 1yph, so we’ll be looking for the histidine and aspartate members of the triad.

Below the Jmol window in the browser, there is a box where you can enter Jmol Scripts. You just type in the command, then click on the Jmol Script button. Enter “select his57”.
Enter “color red”.
Enter “wireframe 100”. This will bring up the catalytic histidine from 1yph. It may bring up the catalytic histidine from the other structure. If it does not, you will need to be creative. Here is one command that may help, but you will need to know the chain identifier for the second structure. If the chain for the second structure is a, then try this command: select his and *a;color yellow;wireframe 100. Then identify the histidine in the second structure that aligns with his 57 in 1yph.
Enter “select asp102;color yellow;wireframe 100”. You may have to repeat the approach from the previous step to identify the catalytic aspartate.
Enter “select protein;backbone off”. Now zoom in and see how closely aligned his57 and asp102 are in the two proteins.

9.To find the third member of the catalytic triad, repeat steps 4-7 using 1yph chain e and one of the chains from your second structure, looking for ser195 (from 1yph, chain e) and the analogous serine in your second structure.

Structure Alignment - alternatives to consider. Instructors are encouraged to modify this exercise to consider other enzymes of particular interest to them. Another alternative would be to explore the pairwise sequence alignment tools on the PDB Compare Structure site, then compare the findings of from sequence alignment with the finding for structure alignment. This could easily be done with chymotrypsin (1yph).