Aligning Sequence to Template Adding Loops Manually | BIOL 4550, Study notes of Biology

Download Aligning Sequence to Template Adding Loops Manually | BIOL 4550 and more Study notes Biology in PDF only on Docsity! Bioinformatics 2 -- Lecture 8 Aligning sequence to template. Adding loops manually. What an alignment means to a modeler ACDEFG....HIKLMNPQRSTVWY ||:|| || :| | ||||: .CDDFGACDGHIYIM..QQSTVWF target template I should... Add Ala to the N-terminal Cys using energy minimization. Keep the conserved Phe sidechain and backbone. Cut out the four residue insertion and re-connect G to H. Switch non-similar sidechains Y->K. Possibly move backbone. Possibly pick another alignment. Cut and insert two residues, Asn-Pro. Switch similar sidechains F->Y. Keep backbone fixed. Given this alignment... In-class Exercise: adding a loop. Pre-view of this exercise: Do a PDB search. Do an alignment. Make mutations. Find a loop that fits the insertion. Anchor the loop using alignment constraints. Splice the loop into the template. Energy minimize. In-class Exercise: adding a loop. Find “Sequence 2” on the course webpage. Create a file in FASTA format, then File-->Open or copy and paste the sequence into the top window of SEQ: Edit-->Create Sequence Run a database search. Homology-->PDB search. Load 1IGD Homology-->Align In-class Exercise: adding a loop. Notice that the alignment has 3 gaps, each a single letter. One gap is the second position from the end! (That’s the optimal alignment, but it’s not right. Ask me why.) Move the last letter back one to eliminate the gap. (middle-mouse drag) Now you have a C-terminal extension of one residue. Merge the two 1-residue gaps to make a 2-residue gap. (middle-mouse drag) Draw alignment constraints to keep the new positions aligned. SEQ:Edit-->Constrain residues, select aligned pairs to constrain. Mutate non-identical matches in the template (don’t touch the target!) SEQ:Edit-->Protein-->Mutate In-class Exercise: adding a loop. Split your template sequence before the prefix and after the postfix. Delete the middle piece. Move the loop chain to its position. To split the chain, select the residue after the cut-point. mouseover selected residue+Alt-->split chain To delete a chain, select the chain number. mouseover chain number+Alt-->Delete chain To move a chain, if necessary: mouseover chain number+Alt-->Move chain This is what it looks like. Now merge them. select chains. mouseover “Chain”+Alt-->join chains ACDEFGHIKLMN ............INSRTDSEQ .....................PQRSTVWYYY 1 2 3 In-class Exercise: adding a loop. Make additional mutations so that the new loop matches the target sequence. Zoom in on the splice points. Make peptide bonds. Select atoms. Builder-->single bond button Next double-click the N. Set to sp2, ionization state “0” Delete the extra CO2 oxygen if necessary. In-class Exercise: adding a loop. Selection-->Clear Edit-->Hydrogens-->Add hydrogens Compute-->Partial charges Select just the loop residues. Edit-->Potential-->Unfix Selection-->Invert Edit-->Potential-->Fix GizMOE-->Minimizer Try moving atoms around to improve packing. Move opposite charges together. Put hydrophobic atoms together. Etc. Searching the database for good loops indel lengthprefix coords postfix coords Prefix and Postfix coords are superimposed on the database protein, and the RMSD is calculated. Low RMSD fragments are superimposed on the model to check the energy. A course energy minimization may be carried out first. PDB structures are scanned with a windowsize = indel + prefix + postfix database protein coords ... R M S D The Low-RMSD, low-energy loops are saved in a database for use in loop-swapping step. Loop swapping Each indel has a set of conforming loops (low-RMSD and low-energy). Indels are chosen in random order and loops are chosen from the loop set. When all indels have loops, the energy is (optionally minimized and then..) calculated. If it is on of the best energies in the list, the model is saved. Run Homology modeling tutorial. Follow the instructions in th tutorial exactly, except: You may choose minimization=none for faster results, or minimization=coarse for better results. You may set the number of models to 5 for faster results. After running the tutorial. Download Sequence 3 from the course web page. Use the automated method to make a model. Choose your best model. Submit your best (unrefined) model as Exercise 3. After refining it (don’t do this yet), submit it as Exercise 4.