Obtaining the 3D protein structure

The final result of this sección should be a file in .pdb format where we can find the coordinates of every single atom of the protein. That file could be interpreted by bioinformatics programs like Phymol or Chimera and represent the protein in different graphical ways. To obtain it, there are two different paths depending on one fact: has the protein been previously crystallographed?

A structure obtained experimentally exists

We will simply use this proven structure. This will be the most reliable method due to it has been the result of a group's investigation. To check if there is any previous structure we recommend the following simple steps:

• 1. Access the UniProt server and search for the protein of interest.

• 2. Search for the structure description area and check if it has an entry to the Protein Data Bank (PDB) database.

• 3. In the positive case, access that PDB entry and download the file that is needed.

• 4. In case of no entry, the steps explained in the next section must be performed.

, the PDB is a universal database where are the 3D structure of all proteins which have been obtained its structure experimentally. That is, any structure that we find there is quite reliable and proven. However, predictions of structures are not included, owing to this procedure is not experimental.

A structure obtained experimentally don’t exists.

In this case is necessary to do a structure prediction process. Although this is not the optimal way due to we will start to accumulate error, we can obtain very reliable results. Several different methods can be used for this purpose. We recommend the following two and in this order:

• 1. Homology methods: This method looks for sequences of proteins that are included in the PDB that have a high similarity with our protein and adopt its 3D structure. If the result shows a similarity of more than 30% in the entire sequence, we could take for granted the structure. Otherwise, if it gives inferior, we should move on to the next step. For this step we recommend the Swiss-Model server.

• 2. I-Tasser (Iterative Threading ASSEmbly Refinement): This server uses several methods, as they define themselves: “a hierarchical approach to protein structure and function prediction. It first identifies structural templates from the PDB by multiple threading approach LOMETS, with full-length atomic models constructed by iterative template fragment assembly simulations.” This method is currently the most reliable and they have been the winners in predicting structure in the last 6 editions of the CAP tests.

• 3. As an extra option we can mention Robetta: It is an ab-initio simulation method, where it try to create the protein from 0, using physical simulation and gross computational power. It works best for short sequences and can take a long time to get results, but these are very reliable.

Energy minimization.

This last step is completely recommendable whatever it be the origin of the file with the structure. It will be necessary to have the structure in a state as stable as possible. To minimize the structure we can use 2 strategies:

• 1. Quick vacuum minimization This is the simplest method. For this purpose we will use the Chimera program and within its tools we will choose the "Energy minimization". Then we will adjust the parameters to what is most convenient for us.

• 2. Minimization in aqueous medium: Complex method, for which we recommend looking for a tutorial to understand the management of the programs used and the necessary concepts. In this more complex case, we will use the VMD program (Visual Molecular Dynamics). It will be necessary to build a structure around the protein with the "Automatic PSF builder" tool, which includes water molecules and ions. Subsequently a simulation of energy minimization will be carried out with the tool "AutoIMD" which in turn calls an external program (NAND) that must be installed on the PC. Finally, the resulting file should be saved and opened in Chimera to eliminate all water molecules and leave only the structure of the protein.

Figure 1. Comparison of the structure of Ole E1 before (purple) and after (blue) an energy minimization process.

Figure 2. Ole E1 after an energy minimization process represented as ribbon and as ASA (Accessible Surface Area). Made by Chimera tool..

Obtaining the 3D aptamer structure

Cosas del apartado 1, se necesita meter mas divs seguramente

Obtaining the 3D protein structure

Cosas del apartado 1, se necesita meter mas divs seguramente

Obtaining the 3D protein structure

Cosas del apartado 1, se necesita meter mas divs seguramente