Try it! – Align multiple structures using jFATCAT

This tutorial demonstrates how to align two structurally homologous protein files within a single document. This technique is useful for discovering similarities and differences between related structures. The files used in this tutorial will be opened from the RCSB Protein Data Bank (PDB) using the Protean 3D interface:

• 1BFD – The crystal structure of benzoylformate decarboxylase

• 1PVD – The crystal structure of pyruvate decarboxylase

These two proteins exhibit structural homology, although they have only a 19% sequence identity. Their structural homology may indicate either distant sequence homology or convergent evolution.

1. Choose File > Open Group to launch the Open Group dialog.

2. Keep the default setting of Center on-screen to align similar structures.

3. Click the Add PDB ID button.

4. Type 1BFD into the text box. You may initially see a yellow warning icon ( ) prompting you to type in a valid ID. Check the box next to Add another and press OK.

The first structure is added to the Open Group file list.

5. When the Open from PDB dialog re-opens automatically, type 1PVD into the text box. Uncheck the box next to Add another and press OK.

The second structure is added to the Open Group file list. (Note: Blurred areas below denote where the user name appears).

6. Click the OK button.

Now that both structures are open in a single document, we will color them by chain to facilitate their comparison.

7. Double-click on the black background of the Structure view to remove any existing selection.

8. Choose View > Style > Color to open the Color bar. From the Color Style drop-down menu, select Color by chain.

The 1BFD structure now consists of a single green chain, while the 1PVD structure consists of two chains: one blue and the other yellow. Since we have not yet aligned the structures, the three chains overlap randomly.

9. To select the chains you wish to align, open the Molecules section using View > Explorer > Molecules. Hold down the Ctrl (Win) or Cmd (Mac) button and click once on the name of the "A" chain from 1BFD and again on the name of the "B" chain from 1PVD.

*p(banner tip). Note: Checking the boxes in the Molecules section only toggles between displaying or hiding the chains in the views. To select an item, you must click on its name.

10. To align the selected chains, choose Structure > Align Structures > Rigid-body Alignment for Selected Regions (jFATCAT). During alignment, a status bar appears in the lower left corner of the Structure View. The status bar includes a red button that could be used to stop the alignment, if desired in the future.

11. When alignment is complete, double-click on the black background to clear the selection. Observe that chain A of protein 1BFD (green) is now aligned to chain B of protein 1PVD (blue).

12. Open the Details panel using the View > Details command, and look for the Root Mean Square Deviation (RMSD). Note that the RMSD value for the two proteins is 3.079 Å.

RMSD quantifies the degree of similarity by describing the average distance, in Angstroms (Å), between the atoms of two superimposed structures. The RMSD value is inversely proportional to the degree of similarity between the structures.

As a very general rule of thumb:

• RMSD ≤ 0.5 Å indicates near-identical structural similarity.

• RMSD between 0.5-2.0 Å corresponds to a high degree of similarity.

• RMSD between 2.0-5.0 Å indicates a lower, but still noticeable, degree of similarity.

The RMSD in this case is 3.079, which is compatible with our knowledge that the two sequences have a 19% sequence identity.