 Dear students, in this module we will continue to explore the 7 steps in homology modeling. As you already know that these 7 steps need to be performed one after the other in order for you to perform homology modeling. Just to give you a little bit of background again, homology modeling involves predicting the structure of a protein by looking at the protein structures that are already known. So given that some of the structures are known, you look at the amino acid sequences of these known structures and then use them to predict the structure of a protein whose structure is unknown. So the 7 steps in order are the template recognition and initial alignment. This is followed by alignment correction and then the generation of the backbone followed by the loop modeling and rotamer association. This is also called the side chain modeling. Next is the model optimization and then once all of these steps are done you want to validate the model, the structure that you have computed. So let's take a look at alignment correction in this module. So you have performed multiple sequence alignment between all the proteins and arrived at the proteins which are similar to the sequence that your protein has whose structure you want to determine. Okay so let's start. If you have an alignment with you from blast already then you would want to fine-tune this alignment. What does fine-tuning mean? Fine-tuning means that residues such as alanine which are replaceable by glutamine but such a replacement cannot occur if the alanine amino acid is there in the hydrophobic core. So let's take a look at this cartoon here. So this is the external membrane of the protein. So the surface amino acids are located here and inside is the hydrophobic core as shown here in red. So this hydrophobic core for instance if it has alanine here then it is not possible to be replaced by glutamine. However if this amino acid was not in the core but rather at the surface then such a substitution might have been possible. So such fine-tuning of alignment is necessary for you to evaluate the substitution of amino acid such that they are feasible. So you can use multiple sequence alignment tools such as Clustol W to find residues and properties that need to be conserved. So by looking at the conserved properties you can make sure that amino acids that are giving rise to those properties are conserved and therefore not substituted. And lastly you examine the template structure to check which residues are in the core hence less likely to change than the residues on the surface. Please remember that the protein is a three-dimensional structure and the surface of the protein has those amino acids which evolve and therefore are substituted more frequently as compared to those amino acids which are there in the core of the protein. Next insertions and deletions that is insertion of an amino acid within a sequence so for instance if you have an amino acid sequence like that so you need to have an insertion here in order to bridge this gap or if you have an amino acid that is extra then you can have a deletion here as well. So by deleting it you are connecting the backbone back again. So these two things can only be done in regions of the protein which are highly variable. This is very important to note that insertions and deletions can only be done where variability in the protein sequence and structure is welcomed. So multiple sequence alignment can help you to find these regions. Next step is to shift the gaps such that the gaps are as small as possible. So here if you have a gap as you can see there is a gap here. So these amino acids need to be removed so you cut the protein here and here and therefore you have removed certain amino acids and then you join these two together to give rise to a new protein that is slightly shorter in length. So once you have removed the amino acids that were there that were extra you can see that there is a gap that is here now. So you need to cater for these gaps as well after you have deleted the extra amino acids. So for that you need to optimize how this gap will be placed in the structure. So as I just mentioned that the gap needs to be as small as possible. So here if you look very carefully in this example so the first situation is that the red line here describes the protein structure in such a way that the distance between this residue and this residue is very large. So this distance is large. Now we want to manipulate the structure such that this large distance starting from here till here it becomes small. So how can it become small you simply shift these two residues here and here. So you have just taken two residues from this portion this segment of the structure and shifted them to the other side of the structure. So essentially there is no change in the sequence just that two residues from the right fragment have been shifted to the left structural fragment. So what does it lead to let's see. So now the blue structure is the new one. So as you can see we cut the protein from here the structure and now the distance between the two amino acids is small as compared to the distance that existed before. So this was large and now it is small. So this is how you reduce the distance between the amino acids in the structure and therefore reducing the distance in the gap. So in conclusion the structural prediction now stands at a place where gaps have been minimized and therefore you are ready to move to the next step that is to assemble the backbone.