FIGURE 1.
Mapping contacts of the lateral gate of SecY by in vivo cross-linking. A, cryo-EM structure of the E. coli SecYEG complex (3) (Protein Data Bank codes 3J00 and 3J01); the four TM domains that constitute the lateral gate are highlighted and the residues where pBpa was incorporated for cross-linking in vivo and in vitro are indicated. B, functionality of pBpa-containing SecY derivatives was analyzed by expressing them in the cold-sensitive E. coli SecY39 strain that contained the orthogonal aminoacyl-tRNA synthetase/tRNACUA pair. Cells were grown overnight on LB medium, diluted, and grown for another 2 h before serial dilution. Of each dilution, 10 μl were spotted on either LB plates (−pBpa) or on LB plates containing 1 mm pBpa (+pBpa). Cells were grown overnight at either 25 or 37 °C.