Computational protocol: The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola

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Protocol publication

[…] Large and small nanodiscs were prepared using MSP1E3D1 and a truncated construct (D7) derived from MSP1D1. DNAs encoding both constructs were cloned into pET28a for expression in E. coli BL21 (DE3) and purified using the protocol developed by Sligar’s group. Nanodiscs were formed in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC; Avanti Polar, USA) lipids at RT. D7 or E3D1 were mixed with refolded MOSPC at a D7/E3D1-to-MOSPC ratio of 2:1. DMPC lipids were then added in a molar excess of 20 and 100 for D7 and E3D1, respectively. The reaction mixture was kept on a shaker for 1 h, after which SM2 Bio-Beads (Bio-Rad, USA) were added to the mixture and left gently suspended overnight. Beads were filter-separated from the reaction mixture and further subjected to SEC on S200 column (GE, USA) equilibrated with Tris-NaCl buffer at pH 7.5. Peak fractions containing MOSPC incorporated into the discs were used for transmission electron microscopy (TEM), performed at the Biosciences Electron Microscopy Facility of the University of Connecticut. Discs containing MOSPC were diluted several-fold in water to obtain a well-dispersed population, applied to a glow discharged, carbon-coated, 400-mesh copper grid (Ted Pella Inc., USA), and negatively stained with freshly prepared 0.75% uranyl formate (SPI-Chem, USA). TEM images were taken on a Tecnai G2 Spirit BioTWIN microscope (FEI, USA) at an accelerating voltage of 80 kV with a defocus of ∼−1.6. Refolding efficiency was assessed through far-UV CD spectroscopy; images of the nanodiscs were obtained by transmission electron microscopy (TEM) and averaged over 70–80 discs/image using the Xmipp software suite. [...] Mid-late logarithmic-phase T. denticola were encapsulated in low-melting-point agarose microdroplets, and washed extensively with DMEM (Thermo-Fisher, USA) prior to the addition of primary antibodies. Beads then were resuspended in DMEM with 2% BSA and 1:200 dilutions of rat antisera against MOSPN, MOSPC, or T. denticola periplasmic flagellar filaments in the presence or absence of 0.05% (v/v) Triton X-100. After overnight incubation with gentle mixing at 4 °C, beads were washed three times by low-speed centrifugation (500 × g) and incubated for 2 h at RT with DMEM containing 2% BSA and 1 µg/ml of goat anti-rat Alexa Fluor 488. Following further washing with DMEM, the gel microdroplets were mounted onto glass slides with Vectashield® anti-fade reagent (Invitrogen) containing DAPI. Fluorescent images were acquired on an epifluorescence Olympus BX-41 microscope using a 100X (1.4 NA) oil immersion objective equipped with Retiga Exicharge-coupled-device (CCD) camera (Q Imaging, USA) and DAPI and fluorescein isothiocyanate (FITC) filter set. The data were analyzed using Cell M (Olympus) and ImageJ. [...] Non-redundant database searches were performed using BLASTP. Conserved protein domains were identified using NCBI CDD-Search. Proteins used for phylogenic analysis of TDE1658 were obtained from the UniProtKB database using the search terms “SurA” and “PrsA” and filtered to include only reviewed records. Closely-related sequences (>65% identity) were removed using CD-HIT. The list of selected sequences was refined further using a guide tree generated in Clustal Omega to remove orthologues for the ribosome-associated chaperone Trigger Factor, which formed a separate cluster. Putative SurA/PrsA orthologs from Treponema sp. and Borrelia burgdorferi were obtained from UniProtKB. The Leptospira interrogans SurA ortholog was identified by Giuseppe et al.. Multiple sequence alignment of the sequences was performed using MUSCLE with default parameters. Phylogenetic analyses were carried out using the PHYLIP 3.696 package. Pairwise sequence distance matrix was computed using Henikoff/Tillier Probability Matrix from Blocks (PMB) matrix in ProtDist and trees were constructed using Fitch with global rearrangements. Confidence levels for the bifurcating branches were obtained using Seqboot program with 1000 step bootstrapping. Phylogenetic trees were visualized by using iTOL. […]

Pipeline specifications

Software tools Xmipp, ImageJ
Applications cryo-EM, Microscopic phenotype analysis
Organisms Treponema denticola, Escherichia coli