24  Comparison of Structure Models with Reality

Author
Affiliation

Dr Randy Johnson

Hood College

Published

October 22, 2025

Introduction

During this in-class exercise we will be comparing the results of structure models of a protein with crystal structures. Specifically, we’ll be working with the Hen Egg White Lysozyme (HEWL) protein in chickens and homologs in several different species.

Setup

Species

We’ll be working with the following species today. Pick one and follow along as we explore these different C-type Lysozyme homologs together.

Bird Species Sequence/Structure Availability Comparison
Chicken (Gallus gallus)

PDB Structure

UniProt

 Reference: This is the protein we will use as a homology target
Guinea Hen (Numida meleagris)

PDB Structure

UniProt

 Very High Homology: Differs from HEWL by only about 10 amino acid substitutions, making it an excellent very close homolog to see minimal changes.
Quail (Coturnix japonica)

PBD Sructure

UniProt

 High Homology: Another close relative within the Galliformes order, useful for subtle comparison.
Duck (Anas platyrhynchos)

PBD Structure

UniProt

 Moderate Homology: Divergence is greater than guinea hen, showing the effect of slightly lower sequence identity on modeling accuracy.
Chachalaca (Ortalis vetula) UniProt Distant C-type: Sequence analysis shows it is surprisingly divergent from other Galliformes like chicken, offering a good test case for homology modeling limits.

Instructions

Reference Identification

  • Pull the amino acid sequence of your chosen species from UniProt

  • BLAST your sequence against chicken (Gallus gallus)

  • Pick the best match and copy the alignment into a text editor to clean it up

Cleaning up the alignment

SWISS-MODEL isn’t going to like numbers and extra stuff in the alignment, so we first need to convert it to fasta format. If we have something like the following:

Query  1    MRSLLVLVLCFLPLAALGKVYGRCELAAAMKRHGLDKYQGYSLGNWVCAAKFESNFNTQA  60
            MRSLL+LVLCFLPLAALGKV+GRCELAAAMKRHGLD Y+GYSLGNWVCAAKFESNFNTQA
Sbjct  1    MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQA  60

Query  61   TNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDV  120
            TNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSD 
Sbjct  61   TNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDG  120

Query  121  HGMNAWVAWRNRCKGTDVNAWIRGCRL  147
            +GMNAWVAWRNRCKGTDV AWIRGCRL
Sbjct  121  NGMNAWVAWRNRCKGTDVQAWIRGCRL  147

We’ll need to copy/paste the Query line (this is our target sequence, i.e. the sequence you are trying to model) onto the first line of the fasta, then the Sbjct line (i.e. the template sequence for chicken) onto the second line. It should end up looking something like this:

>Quail;Target_Lysozyme
MRSLLVLVLCFLPLAALGKVYGRCELAAAMKRHGLDKYQGYSLGNWVCAAKFESNFNTQA
TNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDV
HGMNAWVAWRNRCKGTDVNAWIRGCRL
>Chicken;Template_Lysosome
MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQA
TNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDG
NGMNAWVAWRNRCKGTDVQAWIRGCRL

Homology Model

  • Build a homology model on SWISS-MODEL
    • Enter your cleaned up alignment into the “Target-Template Alignment” section
    • It may tell you that there are multiple biounits and / or chains that were found. This is common for well studied proteins. Use 4cj2.1.A as this is the HWEL
TipChosing a template

If you only have a sequence to work with, you can enter the sequence and use the template finder in SWISS-MODEL to identify an appropriate template. Since we want to compare results across these different species, we’ll all use the HWEL template above, but there will likely be others that are better matches for more distant species.

Alphafold

Next go to Alphafold and use it to search up the model for your chosen sequence. Since all our species have UniProt accession IDs, we can use those directly.

Compare results

Lastly, let’s compare the different structures.

  • Download pdb files for each structure

  • Open PyMOL2

  • (optional) Change directories to the location of your pdb files with something like os.chdir("c:\\Users\\johnson\\Downloads")

  • Load your pdb files with something like load 5YIN.pdb

  • Compare them using something like align 5YIN, AF-P00698-F1-model_v6

  • Export an image and submit it on Blackboard