Homework

Links:http://docs.google.com/spreadsheets/d/1AsYRLlrRLd6I8abxNHfuz1OtFTSqYZ87_kefBMsxhMo/edit?gid=0#gid=0https://docs.google.com/spreadsheets/d/19_u8Sd8TdseHP6yAVrDSjIKf0vDU9RNH3SEACx8L22Y/edit?gid=0#gid=0

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Objective:

1. Learn basic concepts: amino acid structure, 3D protein visualization, and the variety of ML-based design tools.
2. Brainstorm as a group how to apply these tools to engineer a better bacteriophage (setting the stage for the final project).

Part A. Conceptual Questions

• Answer any of the following questions by Shuguang Zhang:

  • How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons)

  500g meat * (0.2 grams of protein / 1 gram of meat) * (6.022x10^23amu/1gram) * (1 amino acid / 100 amu) = 6.022x10^23 amino acids

  • Why humans eat beef but do not become a cow, eat fish but do not become fish?

  Because we digest the macromolecules that we ingest

  • Why there are only 20 natural amino acids?

  The system evolved to be most efficient while allowing to code for many different proteins as well as being stable given the environmental conditions.

  • Can you make other non-natural amino acids? Design some new amino acids.

  Yes. You can make non natural amino acids. I drew one in my lab journal

  • Where did amino acids come from before enzymes that make them, and before life started?

  from outer space. Solar rays will inevitably, randomly make them

  • If you make an alpha-helix using D-amino acids, what handedness (right or left) would you expect? left handed
  • Can you discover additional helices in proteins?

  any times you have designs that attract each other in a pattern, it could form a helix

  • Why most molecular helices are right-handed?

  becuase the underling chirality of amino acids is L amino acids. With L amino acids, right handed helix are more stable

  • Why do beta-sheets tend to aggregate?

    • What is the driving force for b-sheet aggregation?

    hydrogen bonds at the edges of the sheet

  • Why many amyloid diseases form b-sheet? beta sheets form intermolecular hydrogen bonds that are highly stable, and so have a hard time being broken down

    • Can you use amyloid b-sheets as materials?

    Yes, since they are highly stable and structures

  • Design a b-sheet motif that forms a well-ordered structure.

  YMNWQFNAQFYMNWQFNAQFYMNWQFNAQFYMNWQFNAQF

Part B: Protein Analysis and Visualization

In this part of the homework, you will be using online resources and 3D visualization software to answer questions about proteins.

1. Pick any protein (from any organism) of your interest that has a 3D structure and answer the following questions.

   • Briefly describe the protein you selected and why you selected it.

   I am interested in MS2g2, the coat protein for the MS2 Phage. 180 of these proteins come together to form an icosahedron shell. I am interested in this protein because it overlaps the lys protein of the MS2 phage and so any mutations of the former could potentially affect the latter.

   • Identify the amino acid sequence of your protein.

   Using UniProt, I found the Sequence:

   MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY

   • How long is it? What is the most frequent amino acid? You can use this notebook to count most frequent amino acid - https://colab.research.google.com/drive/1vlAU_Y84lb04e4Nnaf1axU8nQA6_QBP1?usp=sharing*

   The sequence is 130 amino acids long then plugged the sequence into ProtPram to find the amino acid breakdown. The breakdown is shown below. The most common amino acid is a tie between Alanine and Valine, at 14 amino acids each.

   

   • How many protein sequence homologs are there for your protein?Hint: Use the pBLAST tool to search for homologs and ClustalOmega to align and visualize them. Tutorial Here

   There are 111 sequence homologs in total, I took a screenshot of part of them. As this virus is so small, its coat protein is highly efficient, and so it is almost entirly conserved along its length.

   

   • Does your protein belong to any protein family?

   The protein belongs to the Leviviricetes capsid protein family.

   • Identify the structure page of your protein in RCSB

     • When was the structure solved? Is it a good quality structure? Good quality structure is the one with good resolution. Smaller the better (Resolution: 2.70 Å)

     The structure was solved in 2005. The resolution is 2.68 Å and so is a good quality structure.

     • Are there any other molecules in the solved structure apart from protein?

     There are no other molecules in the solved structure apart from protein

     • Does your protein belong to any structure classification family?

     [To Do]: Having trouble with this. No structure classification family found

   • Open the structure of your protein in any 3D molecule visualization software:

     • PyMol Tutorial Here (hint: ChatGPT is good at PyMol commands)
     • Visualize the protein as "cartoon", "ribbon" and "ball and stick".

     

     

     

     • Color the protein by secondary structure. Does it have more helices or sheets?

     The protein has more sheets

     

     • Color the protein by residue type. What can you tell about the distribution of hydrophobic vs hydrophilic residues?

     The hydrophilic and hydrophobic residues are evenly distributed

     

     • Visualize the surface of the protein. Does it have any "holes" (aka binding pockets)?

     There doesn’t appear to be any “holes” which makes sense, as this is a coat protein. Or maybe there are holes that neighboring subunits fit into.

     

Part C. Using ML-Based Protein Protein Tools

<aside> <img src="/icons/snippet_lightgray.svg" alt="/icons/snippet_lightgray.svg" width="40px" /> Resources:https://colab.research.google.com/drive/1hXStRY9VCyw52n17uWdWQBj__IcR2ztK?usp=sharing#scrollTo=38gFJBazNdzJ

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• Fold your protein with AlphaFold or ESMFold or Boltz and compare it to the real structure.

• Comment on:

  • Any predicted vs. experimental differences.

  The protein structure is very different compared to the experimental structure. THe biggest difference is that it is a lot more compact. I am not sure why the large disparity

  • Low-confidence regions and why do you think they are low confidence?

  Almost the entire protein structure has a low confidence. This is pretty self evident, as the structure is not the same. Perhaps I have input the incorrect sequence. I will do further research.

  Update. It looks like the simulated structure aligns closely with the upper half of the experimental structure. Im not sure why there is a discrepancy between the number of amino acids in one vs the other.

  Update 2: The pdb file that i Used for the experimental was composed of 3 polypeptides and aparantly 2 ligands???

Structure pulled from RCSB: