Obtaining the 3D protein structure

The final result of this section 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 graphically. 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 being 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.

Clarification, 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 start with a structure prediction process. Although this is not the optimal method because we could 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 be certain the structure. For this step we recommend the Swiss-Model server. Otherwise, if it gives an inferior value, we should move on to the next step.

• 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 mention Robetta: It is an ab-initio simulation method, where it tries 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 reliable results.

Energy minimization.

This last step is completely recommendable whatever the origin of the file with the structure be. It is necessary to have the structure in a state, as stable as possible. To minimize the structure we can employ 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

Previously there were two options, that the protein had a known structure or not. In this case it is a DNA chain that we have personally obtained in the laboratory, so the possibility of find a structure published for that exact sequence it's almost zero. In this way the only case to consider is to try to predict its structure. This action presents a series of drawbacks nowadays. While for proteins there are numerous methods and servers with high reliability, for nucleic acids there are hardly any servers and prediction methods. This is due to the structure of proteins has always been very important and varied for its function, but from the genetic material, the important thing has always been its code, whether in the form of DNA or RNA. For the case of RNAs, there is more than one function, reason why in certain occasions if it has been attended to the need to know their structures but it has not been developed correctly. Even so, we propose a series of steps to predict the structure of the aptamer. Although this predicted structure will not be reliable and it is important to understand this, it can serve us for our objective of proposing an improvement in the initial sequence.

Step 1: Obtaining the secondary structure

As there is no single server as in proteins to obtain a final result giving only the sequence, we have to divide the process in different stages. In this first stage we need to obtain a secondary structure of the single strand of DNA. The server we recommend is the MFold server of the University of Albany. It provides both a graphic map of the links between bases, and the secondary structure in text format (in the Vienna format that we need later), as we show in this example:

GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC
(((((((.................)))))))

Figure 3. Example image of the links between base pairs that the server provides.

Step 2: Prediction of 3D structure

The next step is to predict the tertiary structure. The only server capable of doing that nowadays is ROSIE and it requires the secondary structure that we have previously obtained in Vienna format in addition to the sequence. This server predicts RNA structures (not DNA), so it will be necessary change the T for the U in the sequence.

The result obtained will be a series of three-dimensional RNA structure proposals in .pdb format, so a posteriori it will be necessary to make file modifications to eliminate the excess atoms and modify the links so that it can be identified as a DNA chain.

We must take into account that the algorithm used by this server is not as tight as in proteins and usually commit some deviations in the ab initio simulation. It is particularly very important to know if our aptamer has incorporated some ion, because this method is not taken into account by aptamers that incorporate external molecules and in those cases it is usually deviated a lot in the final result. .

Subsequently it will be necessary to perform a process of energy minimization as explained in the previous section, choosing the method that is preferred to have the most stable molecule.

Figure 4. Trombine aptamer structure comparison between the crystallographed in a lab (blue) and the prediction one (grey). We can see that the blue one have a ion that conditions the structure.

Fact: We tried to fine-tune this step with an aptamer already characterized of thrombin. Although, theoretically, the protocol was logical, the results obtained with the set-up led us to conclude that we did not have it prepared yet due to the high error rate.We tried to fine-tune this step with an aptamer already characterized of thrombin. Although theoretically the protocol was logical, the results obtained with the set-up led us to conclude that we did not have it ready yet due to the high error rate.

Docking procces and mutation proposal

After obtaining the structures of the protein and the aptamer, we need to know in which area and with which orientation the interaction between them takes place; Studying which pairs of bases and which amino acids are involved in this interaction and propose a change of 1 or 2 bases in order to make this interaction stronger.

Docking

The docking is the computational simulation method in which the ideal orientation of an organic molecule is calculated when interacting with another one forming a stable complex (if it exists). Generally this process is designed for protein-protein interactions, reason why most servers are optimized for these cases. However, we recommend using the NPDock server. This server allows the simulation of the interaction between a protein and a single-stranded nucleic acid molecule, having to identify whether it is DNA or RNA before sending the work.

Figure 5. Interaction between thrombine and an aptamer simulated by the server NPDock.

Proposal of mutation

With the .pdb file obtained from the previous step, we only have the most subjective step. In this case we have to perform a study of the interaction and see which atoms interact with each other. For this, the most useful thing is to use a visualizer like the ones we mentioned at the beginning (Chimera or phymol). With the evaluation of the different options will have to interpret if making any change in the aptamer, these interactions could improve

The proposed mutation cannot be accepted blindly. It will be necessary to synthesize the proposed aptamer and check in the laboratory if its affinity is superior to that of the original aptamer.

Links of interest

• UniProt:Database with all proteins and the related information.

• PDB:Database with all 3D shapes of protein, nucleic acids, and complex assemblies.

• Phymol:Is a user-sponsored molecular visualization system on an open-source foundation, maintained and distributed by Schrödinger.

• Chimera:A highly extensible program for interactive visualization and analysis of molecular structures and related data, including density maps, supramolecular assemblies, sequence alignments, docking results, trajectories, and conformational ensembles.

• VMD:A molecular visualization program for displaying, animating, and analyzing large biomolecular systems using 3-D graphics and built-in scripting.

• Swiss-Model:A fully automated protein structure homology-modelling server, accessible via the ExPASy web server, or from the program DeepView (Swiss Pdb-Viewer).

• I-tasser:A hierarchical approach to protein structure and function prediction.

• Robbeta: Ab-inition full chain protein structures prediction server.

• Albany University MFold: Web server for the prediction of secundary structures in DNA single-strand chains.

• Rossie: Web server for ab-initio RNA single-stand structure prediction.

• NPDock: Web server for modeling of RNA-protein and DNA-protein complex structures.