There are clearly far too many results here to make sense of, so we will refine the query and reduce the number of hits. Click Refine the results of this search, and you will see a new search page, much like the first one, where you can enter additional search values.

C-type lectins bind predominantly mannose, so we will find all lectin structures that have a molecule of mannose bound to them. Enter "mannose" in the field Associated small molecule field. Also, since we would like to see what molecules these lectins bind, check the List of HET groups button in the Results field on the right of the page. Hit Start search again.

The new list of hits should contain about 50 results - much more manageable.

The role of calcium in mannose binding

Look closely at the list of hetgroups that are bound to the lectin structures in the results list. Many of the lectins that have mannose bound also have calcium bound.

Find the result line for the entry 2MSB and click on the ID code link on the left of the line. This will take you to the PDBe atlas page for the entry:

Look through the various pages in the atlas pages for 2MSB. These pages contain as much information as possible about the structure, sequence, ligands, citations, etc. for this entry. There are also links to various related databases, such as SwissProt and PubMed.

The mannose binding environment in C-type lectins

In the Ligand page of the 2MSB atlas pages, there are schematic diagrams of the various heterogens that are bound to the molecule. You can see the environment in which each ligand is found, by clicking the link marked View the interactions of MAN with 2msb.

The MSDsite database catalogues the interactions between ligands in the PDB and the protein to which they are bound. You can see all of the bonds, non-bonding interactions, etc. that mannose makes with the protein, and can upload your own structure and use the system to compare it against the whole PDB:

It is interesting to note that the majority of the residues which are used to bind the mannose in this particular C-type lectin are acidic. This is another characteristic of C-type lectins.

Other types of lectin, including other mannose-binding lectins use tyrosine, tryptophan or histidine in the sugar binding site. You can see the different binding environment characteristics of another lectin, concanavalin-A (CON-A) by looking at the MSDsite entry for PDB entry 1cvn:

You can look at the binding sites for the various ligands of these two entries by clicking on the links to the left of each site. The two leftmost icons link to different viewing programs:

	AstexViewer@EBI-MSD - this is a simple java applet that should work on any system which has java installed
	RasMol - this requires your machine to have RasMol already installed (may not work on all machines).

You can also look at the statistics for each type of site throughout the PDBe database, by clicking on the small chart icons next to each hit. The red chart shows the statistics in the database for each kind of ligand, whilst the blue chart links to the statistics environment that are similar to the one which surrounds this particular instance of a ligand. The bonds link shows you the details of the interactions between the ligand and the protein.

Now let's look at what other ligands can be found in similar binding environments. Go to the top level of the MSDsite system. Choose the link marked with a blue chart, showing Ligand Environment statistics. Enter the following environment description into the the Environment field and hit Search statistics:

This search will return a graph showing the statistics for the various ligands which have been found in the specified environment in the PDBe database. You can see that some of the predominant ligands are mannose (MAN), fucose (FUC) and N-acetyl-D-galactosamine (NGA). These are the same ligands that are recognized by most C-type lectins.

So, we started this search by looking at the structures of mannose-binding lectins, and now, by using the ligand binding environment from a mannose-binding lectin, we have found the various other kinds of ligands that other C-type lectins can bind.

Selectivity in binding sites

Now we know that some C-type lectins bind mannose. In fact, we have found that these lectins bind MAN, which in the PDBe database represents the structure of alpha-D-mannose. The database also contains the structure of beta-D-mannose, with the three-letter code BMA.

MAN	BMA

Using MSDsite we can get the statistics for the binding environments for both of these ligands. From the top level of the MSDsite system, choose the Ligand Binding link, to take you to the appropriate search form. Enter "MAN" in one Ligand Field and "BMA" in the other field and press Search statistics button. You will see a chart that shows that the predominant residues in the environment of MAN are ASN, ASP, GLU, SER and THR.

We have looked at several C-type lectins that bind mannose, and which have a role in the innate immune system. Are there any other C-type lectins, or lectin-like proteins, that play a different role in biology ?

To answer this question we will use the MSDfold service. MSDfold searches for structurally similar proteins in the PDB, with respect to their secondary structure. Go to the MSDfold home page and press the Submission button to get to the submission form.

MSDfold can search using a structure that you upload from your own machine, or using any structure in the PDB. We will use the existing PDB entry 2MSB as a search model for MSDfold.

The two most important parameters in the MSDfold submission form are the values of the lowest acceptable match. For now, we will set these values to 60%. The other items in the form can remain unchanged. Enter these values and press Submit your query:

MSDfold can take upto several minutes to run, depending on the size of the search molecule and the matching percentages that are chosen. Whilst it runs you can see the number of CPUs that the system is using to run your particular query. Once the search is complete, you will be taken automatically to the results page:

The most meaningful value in the results table (for our purposes) is the Query % sse. This shows the percentage similarity level between the target structure and the various results. In order to get the results into a more useful order than the default, scroll down the page to find the resort results link. Use this pulldown to find Seq % - the percentage sequence similarity between search and target molecules. The first few pages of the MSDfold results will now contain structures that have very similar sequences to the search molecule, so skip over the first few pages of the results until you find hits where the sequence identity drops to around 60%:

Look down the results and click on the link in the result line for PDB entry 1B08, the structure of lung surfactant protein D (SP-D). If you have RasMol installed, you can look at the superposition of this result with the search structure (the mannose-binding lectin, 2MSB). Alternatively, you can look down the table of results for this match and see the match schematically:

The result protein has a very similar function to that of the search molecule: lung surfactants are found in the epithelium of the lung tissue, where function to bind inhaled pathogens before they can reach the blood stream. However, the sequence similarity between these two molecules is relatively low - only 47%.

Looking further down the result list, we can find other structure which have high structural similarity to the mannose-binding lectin, but rather low sequence similarity. Some examples are macrophage mannose receptor (1EGG) with 26% sequence identity, eosinophil major basic protein (1H8U) with 20% sequence identity, human lithostathine (1LIT) with 19% identity, and flavocetin-A from the habu snake venom (1C3A) with only 17% sequence identity. These proteins have very similar structures to the search molecule, mannose-binding lectin, but sometimes very low sequence similarity, and often very different functions.

Using MSDpro to perform complex queries

The MSDpro tool is an advanced search tool that exposes much of the power of the PDBe relational database, and allows you to combine together many of the search systems that you have already used.

Try using the MSDpro tool to build a single query that finds entries that are similar to PDB entry 2MSB, but which do not bind calcium, mannose, fucose or N-acetyl-D-galactosamine (NGA). This will identify those C-type lectins that have different functions to those that we have already looked at. This is an example MSDpro search to do just that:

  

Looking down these results, you should find at least three of the results that we got earlier, including PDB entries 1C3A, 1H8U and 1LIT.