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Electron Microscopy and Single Particle Analysis of a Mycobacterial Preprotein Translocation, ATPase

Protein translocation across the inner membrane of bacteria is facilitated by the Sec system which is composed by the cytoplasmic ATPase SecA and the integral membrane proteins SecY, E and G that form the heterotrimeric complex SecYEG [1]. Another chaperone, SecB, delivers preproteins destined to be secreted by the Sec system to SecA [2]. The crystal structure of Bacillus subtillus SecA was recently solved to 3 Å resolution [3]. The proposed functional oligomeric state is a homodimer although evidence has suggested that monomeric [4] and tetrameric [5] states are possible. The 10 Å electron crystallographic structure of SecYEG [6] further suggests that SecYEG exists as a dimer whilst others had previously reported a tetrameric state [7]. Thus unless the structure of the entire complex of the translocase machine, SecA-SecYEG, is solved one cannot conclude how the active translocon functions. Combining low resolution EM data with the X-ray data can provide a complete picture of the translocation process. Single particle analysis allows structures to be solved to better than 10 Ă without the need for crystallization which can select for oligomeric states that display higher intrinsic symmetries (e.g. tetramers vs. monomers). Here we report the first EM-derived structure of Mycobacterium tuberculosis SecA at 23 Å resolution, which constitutes the first step along the path to establish the translocon structure. The 3D structure indicates that SecA exists as a tetramer in solution with D2 symmetry and measures approximately 140Å x 100 Å. The addition of substrate and analogues thereof (ATP, AMP-PNP) results in major conformational changes that cause the protein to aggregate as monitored by EM. Preprotein localization studies using nanogold labeling are currently under way.