Negative-staining single-particle analysis electron microscopy and 3D model reconstitution.

For the XcpQ_N012 complex, 10-μl drops of the suitably diluted (0.02 mg/ml) sample suspension were placed directly on glow-discharged carbon-coated copper grids (Electron Microscopy Sciences [EMS]) for 2 min. The grids then were washed with 2 drops of 2% aqueous uranyl acetate and stained with a third drop for 1 min. Images were recorded on an FEI Tecnai 200-kV electron microscope operating at a voltage of 200 kV and a defocus range of 0.5 to 2.5 µm, using an Eagle charge-coupled device (CCD) camera (FEI) at a nominal magnification of ×50,000, yielding a pixel size of 4.4 Å. A total of 500 particles were automatically selected from 60 independent images and extracted within boxes of 100 by 100 pixels using EMAN2. The defocus value was estimated, and the contrast transfer function was corrected by phase flipping without further corrections using EMAN2 (e2ctf). All 2D classifications were performed using EMAN2. We used three rounds of reference-free 2D class averaging to clean up the automatically selected data set.

For Trx-XcpQ_N012 and Trx-XcpQ_N012-S210C complexes, 10 μl of suitably diluted (0.05 mg/ml) Trx-XcpQ_N012 complex and 0.02 mg/ml of Trx-XcpQ_N012-S210C samples were treated as described above for XcpQ_N012. A total of 1,660 particles for Trx-XcpQ_N012 and 3,600 particles for Trx-XcpQ_N012-S210C were automatically selected from 118 and 103 independent images, respectively, and extracted within boxes of 100 by 100 pixels using EMAN2. The defocus value was estimated, and the contrast transfer function was corrected by phase flipping without further corrections using EMAN2 (e2ctf). All 2D and 3D classifications and refinements were performed using EMAN2. An initial 3D model was generated in EMAN2 using 15 and 25 classes for Trx-XcpQ_N012 and Trx-XcpQ_N012-S210C, respectively. All particles were used for refinement. Based on biochemical data suggesting that XcpQ_N012 assembles into dimers and dodecamers, we applied C6 and C12 symmetries. The 6-fold symmetry axis offered the sharpest resolution. The EMAN2 autorefine procedure was used to obtain a final reconstruction at 31-Å and 25-Å resolutions after masking and with C6 symmetry imposed for Trx-XcpQ_N012 and Trx-XcpQ_N012-S210C.