In this practical you will be introduced to the CATH/Gene3D websites and servers that will help you in carrying out an investigation into protein structure and function.

We will begin by looking at the structure of a specific protein (FtsA) and how it can be split into its component domains. We will then investigate the superfamily of one of these domains further by looking at the types of biological functions and how the superfamily is distributed across different biological kingdoms. From this you should gain an understanding of both how to carry out this type of investigation and also, from the real example, how related proteins can vary substantially in structure and function and multi-domain context.

FtsA is essential for bacterial cell division and is found in the hypothermophilic bacterium Thermotoga maritima. This tutorial will show you how to use both CATH and Gene3D to explore this protein with the aim of understanding how evolutionary relatives are distributed across other organisms and with what biological processes they are associated. This investigation will be based around the highest resolution structure available for FtsA - (PDB code 1e4f).

First of all, you need to retrieve all the domains present in FtsA. You can use the CATHEDRAL server to do this. The CATHEDRAL server employs a structural comparison algorithm to compare the query structure against known domains in the CATH database, which means you can also use it to try and identify a unknown protein by comparing it with all known structures in CATH. You submit the protein for analysis at CATHEDRAL search page. You can download the PDB file for 1e4f from the PDB server but, in this case, as 1e4f is known to already exist in the CATH database you can enter the code 1e4f directly into the query box. Then wait for the results to arrive and answer the questions below.

For the remainder of this investigation you are going to focus on one superfamily present in FtaA, the Nucleotidyltransferase domain family (CATH code: 3.30.420.40).

The next step is to investigate one of the domains in the CATH superfamily 3.30.420.40 using both CATH and Gene3D.

To find the domain in CATH, enter the domain code (i.e. 1e4f followed by the chain ID and domain ID) into the search box Click Here and look at the result. If you look at the homologous superfamily level (the H-level) you will see that the domain has the code 3.30.420.40. Click on this level to view the structures of other known domains from this homologous superfamily.

This domain is found in a wide variety of apparently very different proteins with differing molecular and cellular functions. Clearly this needs further investigation. Clicking on the domain ids of the structures in this superfamily brings up more information on that particular domain. The Rasmol link () will allow you to view the structure. The PDBSum link at the bottom right hand corner of the page () should provide you with a summary of ligand interactions and other information.

As you have seen so far, structural data can be a very powerful way of viewing a protein. However, the structural data set, as represented by the PDB, is very sparse; for reasons of cost and ease there is far more sequence data, with associated annotation available. It is possible to predict structural domains in sequences by using 'profile HMMs'. For the next section of the practical you are going see how combining structural predictions with the sequence world can provide a rich view of evolution and function change.

We are now going to use the Gene3D resource to explore this superfamily further. Gene3D provides access to more functional information, as well as taxonomic distributions, multi-domain architectures and protein-protein interaction (PPI) data.

We're going to start with the 1e4fT04 domain you have been investigating. You will take other examples from the superfamily later and make observations from Gene3D.

Follow this link to Gene3D. The Gene3D front page consists of several search options, to retrieve different data types (proteins, superfamily summaries, genome summaries, etc.). For now go use the protein retrieval search option by entering '1e4fT' into the search box (or follow this shortcut Click Here).

A page is return which contains a series of tabs containing structural and functional information related to 1e4fT. Compare the HMM-based predictions (CATH_HMM) to the Pfam domain assignments. Pfam families are normally derived from analysis of sequences rather than structures and so can often contain multiple structural domains that commonly co-occur. Click on the second 3.30.420.40 domain (the discontiguous one) and follow links out to CATH, Gene3D. You should also see some functional sub-classification of this domain in the pop-up.

When you're ready, we can investigate other proteins from this superfamily.

Next try 1dkgD (or follow this shortcut Click Here).. From the domain architecture view we can see that the CATH_HMM architectures is quite detailed and complete.

As an example of this group, search Gene3D with the pdb 1bdg (or follow this shortcut Click Here). The protein is a hexokinase in the parasitic worm Schistosoma mansoni. In this section you are going to search a CATH superfamily and restrict the results according to various criteria.

Looking at the domain architecture in the sequence features tab we can see that the protein has two CATH domains, clicking on the CATH domains we see they have different Funfam annotations. Funfams are subdivisions of CATH superfamilies providing functionally coherent groupings. We can retrieve proteins with similar functions (because they have the same Funfam assignments). by clicking the “Click here for functionally similar proteins” link.

For this family you are going to look at interactions. For this you are going to start with the Actin-related protein (arp) with the PDB code 1k8kA. Search this term in the Gene3D (or follow this shortcut Click Here).

Find the interaction with this protein by clicking on the interactors tab.

You can now start to investigate the sub-processes the interactors of this protein are involved in.