Structural analysis of full-length lysine acetyltransferase 7 by cryo-electron microscopy

Abstract

Objective·To analyze the full-length protein structure of human-derived lysine acetyltransferase 7 (KAT7) using cryo-electron microscopy (Cryo-EM) and to obtain the profile information of human-derived KAT7.Methods·The recombinant protein expression plasmid pGEX-4T1-GST-KAT7 was constructed by using the pGEX-4T1 vector and the full-length gene of human-derived KAT7, and the KAT7 protein was expressed in the prokaryotic protein expression system BL21 (DE3). The GST-KAT7 recombinant protein was obtained by using GST affinity chromatography. After removing the GST protein tag with TEV protease, KAT7 was further isolated and purified by HiLoad 16/600 Superdex 75 pg volume exclusion chromatography. The obtained protein samples were identified by Western blotting, and the samples were screened. The protein morphology was observed under negative-stain electron microscopy, and data were collected by using Cryo-EM. The protein particles were selected and the spatial structure of the full-length KAT7 was analyzed with the Cryo-EM analysis software CryoSparc. The MYST structural domain model (5GK9) in the Protein Data Bank (PDB) and AlphaFold prediction model of KAT7 were matched with the generated structural model by UCSF Chimera software.Results·The full-length protein of human-derived KAT7 was successfully purified by affinity chromatography, and high purity KAT7 was obtained by volume exclusion chromatography. After identifying KAT7 by Western blotting, the spatial structure of KAT7 full-length protein was initially resolved by Cryo-EM and single-particle reconstruction techniques, and a preliminary three-dimensional structure model with a resolution of about 10 Å was obtained by three-dimensional optimization. The spatial structure of KAT7 full-length protein was irregular and semi-loop-shaped, and the existing MYST domain model (PDB: 5GK9) can be matched into the C-terminal part of the KAT7 full-length model. The adjusted AlphaFold prediction model can also match the KAT7 full-length structure model.Conclusion·A preliminary analysis of the spatial structure model of full-length protein of human-derived KAT7 is performed by using Cryo-EM.

Keywords

• lysine acetyltransferase
• post-translational protein modification
• acetylation
• cryo-electron microscopy