Pick one method from the following options (step 2 in the pipeline schematic above):

  1. Mutagenesis using Protein Language Models [Easiest One]

    1. Designing these mutants with good computational confidence is hard. It will show you limitations of some of the structure based models. Ultimately you can pick various combinations of mutations and get lab results and then decide to pick the next round of mutations. But this assay won’t be easy to run at scale in this class. So using the information below you can either make a best guess or you can use the strategy Allan was talking about during recitation. Contact Manu or Allan if you need one on one help.

    2. Run this notebook to generate for each position in the amino acid sequence, a “score” for what would happen to the protein if you mutated into another amino acid. It can be positive or negative for the protein. We want to identify possible mutations that are “positive” If you run this notebook - you will see a .CSV file in the sidebar. You can download it and look at it in the google sheets if that’s easier

    3. Use the experimental data here. This dataset contains information about mutants of the L-Protein and their effect on lysis in the lab - 11WzDDNkQDEiqbUSGV0ZCqITGctyNFpD7xnPlhsj2BhE

    4. First check, does the experimental data correlated with the scores from the notebook in (b) ? This should give you a clue on how well these language embeddings capture information about this protein sequence.

    5. Using information about effect of protein mutations at these sites - both the scores and the experimental data in the drive, come up with 5 mutations for each student along with how you came up with them and why you believe they would work. 2 of the variants you submit must have mutations in the transmembrane region (refer to notes above on what amino acid positions these are) and 2 of them must be in the soluble region . Remember that you can also use the pBLAST to see which residues are conserved and not mutate them if you want to.

      One easy way to generate sequence mutations could be to look for residue positions and mutations that are have a positive mutational effect either in the experimental or have a positive score from step 1. And pick a combination of those mutations.

    6. You can utilize Af2_Multimer to generate a Multimeric Assembly you can do this by making your query sequence as. We want to do this because - A running hypothesis for how this protein function is that it assembles to make a perforation in the bacterial membrane. Our TA Ben Arias-Almeida discovered that when we predict the assemble of 8 structures of the lysis protein*,* it indeed reveals a channel-like complex.

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Original Sequence

METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT:METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLL

1. Mutation 1:

MNPTLALAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYGMNEEGWRRAVWEKNMKMIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKGKVFQEPLFYEAPRSVDWREKGYVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSEQNLVDCSGPQGNEGCNGGLMDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV

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2. Mutation 2: