ジャーナル: Sci Rep / 年: 2021 タイトル: Structural insight into Pichia pastoris fatty acid synthase. 著者: Joseph S Snowden / Jehad Alzahrani / Lee Sherry / Martin Stacey / David J Rowlands / Neil A Ranson / Nicola J Stonehouse / 要旨: Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a ...Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.
Focussed mask used for focussed 3D classification of ACP domain density. The mask only covers the region of the map containing ACP density. This mask was not used for sharpening.
モデルのタイプ: INSILICO MODEL In silico モデル: Initial model generated using SGD in RELION 3.0.7.
最終 再構成
想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 3.1 Å / 解像度の算出法: FSC 0.143 CUT-OFF / ソフトウェア - 名称: RELION (ver. 3.0.7) 詳細: Asymmetric reconstruction without alignment was performed on each of two subsets (randomly assigned) from an ACP domain density-containing class after focussed 3D classification. This led to ...詳細: Asymmetric reconstruction without alignment was performed on each of two subsets (randomly assigned) from an ACP domain density-containing class after focussed 3D classification. This led to the generation of two half-maps, which were used to generate a sharpened map from all the data. Note that the number of particles given above includes particles contributing to multiple symmetrically redundant orientations, as particles were generated through focussed 3D classification. 使用した粒子像数: 117012
初期 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.0.7)
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.0.7)
最終 3次元分類
クラス数: 10 / ソフトウェア - 名称: RELION (ver. 3.0.7) 詳細: After D3 symmetrical reconstruction in RELION, focussed 3D classification was performed on the resultant set of particles and orientations after symmetry expansion (i.e., each particle was ...詳細: After D3 symmetrical reconstruction in RELION, focussed 3D classification was performed on the resultant set of particles and orientations after symmetry expansion (i.e., each particle was classified in each symmetrically redundant orientation) with a mask around the ACP domain-containing region of the complex. Classification was performed in RELION 3.0.7, without alignment, and a regularisation parameter ('T' number) of 40. A class containing high-resolution density corresponding to the ACP domain was selected and particles in this class were used for a final reconstruction without alignment.