Overview

Protein folding determines the overall function of the enzymes, so it is important to predict its structure from our primary protein structure. We used Robetta online tool, an online protein structure service evaluated through CAMEO. It allows modelling multi-chain complexes which provide 5 candidate models for RosettaFold

Assumptions

• Most accurate method based on the benchmark
• Multichain modeling provide MSA with chain sequences

Parameters

• RoseTTAFold
• No PDB template needed

Results

For each variant amino acid sequences, there are 5 possible models of folded protein structure. On the right-hand, it has Angstrom error estimate graph. ?

• Variant 1 Results
• Variant 2 Results
• Variant 3 Results
• PDB files are available on UIUC-iGEM github repo

Variant 1

Figure 1: Results of RoseTTAFold of variant 1

Variant2

Figure 2: Results of RoseTTAFold of variant 2

Variant3

Figure 3: Results of RoseTTAFold of variant 3

Comparing variant structures to wildtype structures

We used pairwise Structure Alignment tool from RCSB](https://www.rcsb.org/alignment)

• Variant 1

Figure 4: Structural alignment with 6EQD WT and variant 1

Our first variant has 85% sequence similarity and 83% sequence identity when compared to the wildtype isPETase sequence. Pairwise structure alignment gave this variant a template-modeling score of 0.79, on a scale from 0 to 1, and a structural similarity score of 664.37.

• Variant 2

Figure 5: Structural alignment with 6EQD WT and variant 2

Our second variant has 87% sequence similarity and 86% sequence identity when compared to the wildtype isPETase sequence. Pairwise structure alignment gave this variant a template-modeling score of 0.82, on a scale from 0 to 1, and a structural similarity score of 686.17.

• Variant 3

Figure 6: Structural alignment with 6EQD WT and variant 3

Our third variant has 99% sequence similarity and 98% sequence identity when compared to the wildtype isPETase sequence, the highest values out of all of our variants. Pairwise structure alignment gave this variant a template-modeling score of 0.84 and a structural similarity score of 756.65, the highest scores out of all our variants.

Visualization of PETase candidates

Above are our predicted structures for the three PETase candidates that will be tested in wetlab, each presented alongside the wildtype isPETase sequence and structure for pairwise sequence alignment. Each test presents a sequence similarity percentage, sequence identity percentage, template modeling score, structural similarity score, and the root mean square deviation. For all parameters, our third variant displays the best results, sharing nearly 99% similarities in structure with the wildtype sequence and also possessing the highest structural similarity score and lowest root mean square deviation.

References

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   Accurate prediction of protein structures and interactions using a 3-track network.

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