We input the amino acid sequence of INP_linker_tyrosinase into alphafold2 for structure prediction and set the iteration five times. PLDDT (Per-confidence scores) images of prediction results are shown in figure 3, with blue areas representing the convergence of prediction results and red areas representing the fluctuation of prediction results. alphafold2 achieved convergence in predicting the structure of both INP and tyrosinase, and there was great uncertainty in predicting the structure of the linker. Since the linker is a peptide chain and there is no obvious secondary structure, which leads to uncertainty in structure prediction, which is in line with biological principles.
<div>Figure3. Per-residue confidence scores of INP_linker_tyrosinase.</div>
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  In addition to PLDDT, PAE (Domain position confidence) is also a common evaluation indicator. Predicted PAE gives a distance error for every pair of residues It gives AlphaFold's estimate of position error at residue x when the predicted and true structures are aligned on residue y. Values range from 0 - 35 Angstroms. It is usually shown as a heatmap image with residue numbers running along vertical and horizontal axes and colour at each pixel indicating the PAE value for the corresponding pair of residues. If the relative position of two domains is confidently predicted then the PAE values will be low (less than 5A) for pairs of residues with one residue in each domain. The blue region representing the two proteins and linker can be clearly seen on the PAE image, which supports the accuracy of the prediction results.
<div>Figure4. Domain position confidence of INP_linker_tyrosinase.</div>
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  By comparing the predicted fusion protein structure with the structure of 5M6B, it was found that tyrosinase and 5M6B had obvious overlap, and the RMSD was 0.663. This result indicated that the structure and function of tyrosinase could not be changed by anchoring to the cell membrane with INP fusion expression.
