7FAO

Top7 surface mutant K42A Q43A K46A K57S K58S, and I68R

Summary for 7FAO

• Entry DOI: 10.2210/pdb7fao/pdb
• Descriptor: Top7 Surface mutant, MAGNESIUM ION (3 entities in total)
• Functional Keywords: top7, surface mutant, de novo protein
• Biological source: unidentified
• Total number of polymer chains: 2
• Total formula weight: 21576.15

Authors

Ito, Y.,Makabe, K. (deposition date: 2021-07-07, release date: 2022-05-18, Last modification date: 2023-11-29)

Primary citation

Ito, Y.,Araki, T.,Shiga, S.,Konno, H.,Makabe, K.
Surface Engineering of Top7 to Facilitate Structure Determination.
Int J Mol Sci, 23:-, 2022

PubMed Abstract: Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/R values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

PubMed: 35054886
DOI: 10.3390/ijms23020701

Experimental method

X-RAY DIFFRACTION (1.43 Å)