 Dear students, in this module we will continue to look at the 7 steps in homology modeling. At this point, you have already performed sequence alignments and shifted the residues such that the gaps have been minimized. The insertions and deletions have already been performed and you are ready to look at creating the backbone structure of the protein. So let's generate the backbone. So the first step in generating the backbone is to copy the coordinates from the PDB. As you already have a template that is the structure that is known with you, you can obtain the PDB file from the Protein Databank website and copy each position in the backbone to the target structure. So the target structure is what you want to create. So by starting at each amino acid in the template, you find its corresponding x, y and z from the PDB and then you bring it to the target structure. Let's say here is your template and you want to use it to create a target. So PDB gives you the x, y and z of each alpha carbon. So let's call this carbon alpha 1, carbon alpha 2, carbon alpha 3 and so on. So you have alpha carbon 1, so you have x, y and z. Next you have carbon alpha 2 with its coordinates. So this information is there in your PDB and you can have all the alpha carbons that are here in the templates backbone. So once you have looked at the coordinates, all you have to do is to copy each coordinate and paste them in order. So once you have done this exercise what you will be happy to look at is that you are starting to generate the backbone which is obviously very similar to the template. So by looking at the coordinates of alpha carbons in the PDB you can recreate the backbone in the target. So at this point please note that when the residues are the same that is when the template sequence and the target sequence have let's say alanine at the same position then you can also copy the side chain. So let me explain. So here was your structure for the template and let's say if you had alanine here so it would have its own side chain as would other residues. So if the target has alanine here as well in the sequence then you can copy the side chain coordinates as well. For instance if alanine was not here and there was let's say lysine then of course the side chain would be different than alanine. So you would not copy the side chain in that case. So please remember if the amino acid is the same at that position between target and template you also copy the side chain. So once you have done that please remember that PDB may contain slight offsets and errors so it is useful to use multiple templates at a time, multiple similar templates at a time. So the backbone is generated plus remember along with the backbone you have also put the side chains for those amino acids that were existing in the template and the target sequence at the same positions. So you have the backbone plus the side chains for the common amino acids. Now the point is how do we move to the next position. So the next thing is the loop modeling. So let's take a look at loop modeling. If you remember that there were positions in the sequence from where we deleted or inserted some amino acids. So what happens to such gaps? So let's take a look. So when the target sequence contains a gap. So here you have your template like that and here you have your target. So let's say if your target had a gap here. So in case when the target has a gap then one option is to delete the amino acid from the template. But this will create so if this is the structure of your template and if you delete the second amino acid then it will leave you with a broken template structure. You don't want to do that right? So when the template or the known structure contains a gap there are no backbone coordinates. This will mean that if you delete the second amino acid from the example that I just gave you you will obviously have no coordinates for that from the PTB. So for that this will create a problem and to overcome the target backbone has to be cut to insert newer residues. So if your template has an amino acid that is not there in your target. So you have to cut the target and insert new residues. So how do you insert the new residues here? Which residues are to be inserted? So let's discuss that. So because cutting the target will mean insertion of newer amino acids in that position and that the activity of the protein can change therefore the new residues that you will add can only go into the loops and strands. So please remember this very important point that those residues that are inserted into the target in order to fill the gaps typically they go to the loops. Because the loops are flexible both in length and in structure you can select from multiple possibilities and choose one of the loop structures that is most suitable. So how do you choose the most suitable loop? So one way is to handle the loops such that so this is the same example. So here is one amino acid, here is the other one and there is this break between them. So this is the break, right? The gap. So what you can do is you can look at the protein database for loops which can fill this gap. For instance there can be multiple options. So for example one loop can be like that, some other loop from the protein that a bank can be like that, yet another loop can be like that. So from loop one, two, three and others you have to choose which loop you have to select. So the best way is to choose that loop and plug it into the model. So that is the best loop that you have. So in conclusion once you have formulated the protein backbone for the unknown structure and associated the side chains that are similar between for the residues that are similar between the target and the template. Next is you fill those portions in the backbone that had a gap and to fill those gaps you create two anchors in the backbone and you model which loop best suits that position in the structure. So the next step is to use this backbone and place the side chains for each amino acid that is not similar between the target and the template. Remember that for the similar amino acids the side chains have already been placed.