Difference between revisions of "Team:Uppsala/Model"

One of the first things we noticed when looking at our selected enzymes was that no three-dimensional structures were available and the enzymes were poorly characterized in general, so if we wanted to get a sense of what they might look like, we would have to figure something out. The obvious thing to do, we felt, was to turn to homology modeling.

The idea of homology modeling is to construct a 3D-structure by mapping the amino acid sequence onto a template – i.e. a known structure of a related, homologous protein through sequence alignment. For this to work you need a template with a solved 3D-structure. The quality of the model is determined by the alignment with, and, the structure of the template. Thus, ideally, you would like a high-resolution structure with high sequence identity.

While there are various tools available for homology modeling, such as MODELLER, we choose to use SWISS-MODEL /2/, a fully automated protein structure homology-modeling server (1, 2, 3). We plugged in the sequences of our chosen enzymes (CaCCD2, CsADH2946 and UGTCs2) and started modeling. While the available templates weren’t quite as high-quality as we had hoped, we were confident that they were sufficient to get the job done. We chose our templates and obtained the models detailed in figure 1 and the models along with the quality scoring are summarized in table 1. We could make two immediate observations. The CsADH2946 model seemed the most promising one, quality-wise with GMQE close to 1 and higher QMEAN being better. In addition, looking at the N-terminals (the blue ends in figure 1) we could see that they are stretched out, poking outwards from the protein. This was a good indication that we could put a His-tag at this end, with no complications. Another discovery we made was that the second step enzyme CsADH2946 is most likely a tetramer. This information was helpful when purifying the enzyme and going forth with molecular dynamics.

So we constructed homology models of our enzymes. Are they good models? Are they realistic? There are several measurements that can be made on the models to estimate the answers to these questions. One such measurement is Global Model Quality Estimation (GMQE) and QMEAN (4), but the models are still just a guess of what our enzymes actually look like. To asses the models and prepare them for further research we used GROMACS /1/ to simulate our enzymes in saline water for a total of 100 ns. This lets us assess their stability and obtain new models that are closer to their native conformation which would be the most probable state of the enzymes during the activity measurements in our wet lab.

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