<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links: http://docs.google.com/spreadsheets/d/1AsYRLlrRLd6I8abxNHfuz1OtFTSqYZ87_kefBMsxhMo/edit?gid=0#gid=0 https://docs.google.com/spreadsheets/d/19_u8Sd8TdseHP6yAVrDSjIKf0vDU9RNH3SEACx8L22Y/edit?gid=0#gid=0

</aside>

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)

    • Ans: 1 Dalton is $1.66*10^{-24}$ gram, So:

    $$ \frac{500 grams}{100 Daltons} = \frac{500 grams}{100 * 1.6610^{-24}grams} = \underline{3.01210^{24}\:Molecules} $$

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

    • Ans: The things we eat are dead and digested in our bodies while we are a separate living being with different cells already in a structure.

  • Why there are only 20 natural amino acids?

    • Ans: Even I am curious about this, Lets ask chatgpt: ”Only 20 amino acids are standard because evolution favored a set that was chemically diverse, easily synthesized, and reliably encoded in the genetic code. While many more are possible, expanding the set would require major changes to the genetic and translational machinery—too risky once life was established, so evolution "froze" the system with 20.” ~OpenAI’s ChatGPT (May 28, 2025)

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

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

  • If you make an alpha-helix using D-amino acids, what handedness (right or left) would you expect?

  • Can you discover additional helices in proteins?

  • Why most molecular helices are right-handed?

  • Why do beta-sheets tend to aggregate?

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

  • Why many amyloid diseases form b-sheet?

    • Can you use amyloid b-sheets as materials?

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

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.

     • Ans: I chose Deoxy Human Hemoglobin(PDB id 1A3N**)**, as it is a commonly known protein and medically significant. It is used to transport oxygen to cells in our body allowing us to breathe and function.

   • Identify the amino acid sequence of your protein.

     • 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

       • Ans: Using the Subunit beta of hemoglobin from now on, It is 146 Amino acids long, with Valine being the most common, appearing 18 times:

       

     • 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

       • Ans: A 100 sequences come up when searched for:

         seqdump.txt

         clustalo-I20250528-222515-0706-49990485-p1m.aln-clustal_num

         

     • Does your protein belong to any protein family?

       • Ans: Yes, it belongs to the globin family(Pfam: PF00042). Hemoglobin beta is a member of the larger globin superfamily, which includes other oxygen-binding proteins like myoglobin and various hemoglobins from different species.

   • Identify the structure page of your protein in RCSB

     • Ans: https://www.rcsb.org/structure/1A3N

       

     • 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)

       • Ans: The structure was solved in the year 2000 and published in Acta Crystallographica. The resolution is good, at 1.80Å

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

       • Ans: 1) Heme groups (HEM): Four protoporphyrin IX molecules containing iron (Fe) are present, one bound to each subunit. 2) Water molecules: A water molecule was observed in one alpha chain’s heme pocket at 120 K but not the other.

     • Does your protein belong to any structure classification family?

       • Ans: Globins

         

         

         

   • 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".

       • Ans: Cartoon, ribbon, sticks and spheres in order

         

         

         

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

       • We can see only helices and no sheets

         

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

       • Ans: They are distributed alternatively in the helices of the protein

         

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

       • Ans: Yes a clear hole is present in the centre

         

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

</aside>

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

  

  Predicted Structure

  

  Real Structure from https://www.rcsb.org/structure/1A3N

• Comment on:

  • Any predicted vs. experimental differences.
    • Ans: The coloring clearly differenciates the 4 chains but the folder protein is not colored chain wise, not able to clearly capture the quaternary structure of hemoglobin. Alphafold also doesnt predict non-protein ligands (heme group here)
  • Low-confidence regions and why do you think they are low confidence?
    • Ans: Without modeling the tetramer interface residues show poor packing, increasing uncertainty due to the missing inter-subunit context.

• Inverse-fold your structure with ProteinMPNN

  • What sequence do you get?
    • Ans:
  • Is it the same as the original sequence you folded?

Part D. Group Brainstorm on Bacteriophage Engineering

<aside> <img src="/icons/slide_yellow.svg" alt="/icons/slide_yellow.svg" width="40px" /> MIT/Harvard Students can optionally present this on Wednesday, March 5. Due for all on March 11

</aside>

1. Find a group of ~3–4 students
2. Review the Bacteriophage Final Project Goals:
3. Brainstorm Session
4. Individually put your plan on your Notion page