Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter.

Generation of C. crescentus PAmKate Fusion Strains. McpA–PAmKate. To construct C-terminal PAmKate fusions in a single step under a native locus, we used the vector pYFPC-2 (or under the xylose locus, pXyl-YFPC-2) backbones. The YFP gene was cut out using EcoRI and NheI enzymes, and the resulting backbone was purified by separation on an agarose gel. The PAmKate gene was amplified from Addgene plasmid #32691 ( 39 ) and inserted into the above backbones using Gibson assembly, resulting in the pPAmKateC-2 and pXylPAmKateC-2 vectors. The McpA coding region was amplified with appropriate overhangs from genomic DNA from NA1000 cells and gel purified. The pXPAmKateC-2 vector was linearized by digesting with NdeI, and the McpA gene was inserted using Gibson assembly. The linker used for the McpA fusion was GTLSRPENSNVHRS. PAmKate–PopZ. To construct an N-terminal fusion construct, PAmKate and PopZ genes were PCR amplified with a –GGGSGGGS– linker and cloned into pXyl-YFPC-2 that was digested by NdeI/NheI using Gibson Assembly ( 40 , 41 ). The ligation was transformed into Escherichia coli DH5α cells and selected on LB-kan plates. Sequence-verified plasmid was then transformed into NA1000 cells via electroporation. SpmX–PAmKate. In order to produce the plasmid for the SpmX–PAmKate C-terminal fusion on the native promoter, the region encoding SpmX and the 500 base pairs upstream were amplified from genomic DNA from NA1000 cells and gel purified. This gene was then inserted at the EcoRI site on the linearized pPAmKateC-2 backbone. The linker used for the SpmX fusion was PAGALINMHGTLRSRAPENSNVTRHRSAT. Room Temperature Superresolution Imaging. C. crescentus cells were induced with 0.3% xylose for 3 h (McpA–PAmKate, PAmKate–PopZ) or cultured with endogenous expression (SpmX–PAmKate) and placed on agarose pads. These molecules either form relatively fixed structures or do not move dramatically during live cell imaging; highly mobile molecules are rejected as bad fits. Imaging was performed with a custom epifluorescence microscope. Fluorescence emission under 680 W/cm 2 of 561 nm excitation (Coherent Sapphire) was collected using an oil immersion, supercorrected objective (Olympus PLANON60xOSC, 60×/N.A. 1.4) mounted in a Nikon Diaphot 200 microscope and imaged onto an EMCCD camera (Andor iXon) at 20 Hz. Occasional pulses (150 nm; intensity, 18 nm;