Fig. 3. Preparation of MBP-tagged fatty acid synthase.

A MBP insertion site in ^fusFAS chain is shown. Two eight-residue linkers were used to doubly tether the MBP domain (PDB: 3MBP) to FAS1. B ACP domain deletion strategy is shown. ACP model from PDB 6TA1. Purification of asymmetric FAS with deletion of the ACP domain in (C) FAS2 (i.e., prep 4) and D) ^fusFAS chain (i.e., prep 5). From left to right: schematic of cell expressing the MBP tagged constructs, SDS-PAGE of tandem affinity purification (Ni-NTA pull down followed by twin strep-II tag affinity purification) of the engineered FAS, and 3D cryoEM density maps of the purified complex. One MBP domain is highlighted with red circle in each cryoEM map.