Computational protocol: Molecular mechanism governing ratio-dependent transcription regulation in the ccdAB operon

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[…] Data were collected at the SWING beamline, SOLEIL synchrotron (Gif-Sur-Yvette, France) in high pressure (or high performance) liquid chromatography mode (). The CcdB2-CcdA2-CcdB2 complex was prepared at a concentration of 7 mg/ml in 10 mM Tris pH 7.3, 50 mM NaCl by slowly titrating CcdA2 into an excess of CcdB2. The sample was injected onto a Shodex KW402.5-4F column, which had been pre-equilibrated with running buffer (10 mM Tris pH7.3, 50 mM NaCl) for at least one column volume. The flow rate was 0.2 ml/min and data were collected with an exposure time of 750 ms and a dead time of 750 ms. Buffer data were collected at the beginning of the chromatogram and sample data were collected in the peak area. Data reduction was performed on-line using the FOXTROT software (SWING), while buffer subtraction and data averaging was performed using DATASW (). Further analysis of the resulting scattering curve was performed using the ATSAS package (). The indirect transform program GNOM was used to calculate the particle distance distribution function p(r) (). Ten ab initio shape reconstructions were generated using DAMMIF () and averaged using DAMAVER (). The shown ab initio envelope corresponds to the damfilt model.Based on the N-terminal region of the solution structure of CcdA2 (), encompassing residues 1 until 39 (PDB: 2ADL) and the crystal structure of the complex of CcdB2 with the C-terminal part of CcdA, CcdA37-72 (PDB: 3HPW), a model for the CcdB2-CcdA2-CcdB2 complex (model A) was built, assuming that the CcdA2 α-helices from the two models will extend into one continuous α-helix. The Allosmod-FOXS server (,) was then used to generate 100 structures similar to the input structure. The theoretical scattering curve of these models was then compared to the experimental data using CRYSOL (). [...] All EM images were collected on a JEOL JEM-1400 electron microscope equipped with LaB6 cathode and operated at 120 kV. Images were recorded on 4096 × 4096 pixels CMOS TemCam-F416 (TVIPS) camera. The negative stain data were collected at nominal microscope magnification of 50 000 and corresponding pixel size at the detector of 2.29 Å. The defocus was in the range between 1.2 and 3.0 µm. For the sample prepared at high and low CcdA-CcdB/DNA ratio 382 and 358 micrographs were collected, respectively. A total of 77 cryo-EM images were collected at nominal magnification of 40 000, corresponding pixel size of 2.87 Å and defocus range of 1.6 to 3.0 Å. For image processing, helices were boxed in e2helixboxer (), 2D classes and 3D reconstruction by IHRSR procedure were performed in SPARX (). The hand of the thick helices was determined by the tilt method () (see : Electron Microscopy). [...] First, a model for the 113 bp ccdAB promoter/operator DNA was generated using the 3D-DART server (). The helical axis of the model was aligned to the axis of the EM map. To correctly place the first CcdA2 dimer on the central 5΄-GTATAC-3΄ binding site, the solution structure of CcdA2 in complex with this binding site (PDB 2H3C) () was first aligned with the DNA. Then, the antitoxin CcdA2 from the CcdB2-CcdA2-CcdB2 heterohexamer refined to the experimental SAXS curve was aligned to this CcdA2 dimer. The helical symmetry of the 3D reconstruction was then applied to CcdA2 to generate the adjacent antitoxin on the DNA. To connect the two antitoxins, the crystal structure of CcdB2 in complex with two CcdA peptides was used (PDB 3G7Z) (). The CcdA α helices in the latter structure were aligned with the CcdA2 α helices from the CcdB2-CcdA2-CcdB2 heterohexamer. The minimal helical unit for the protein chain finally consists of residues 1–40 of the two central CcdA antitoxin monomers from the CcdB2-CcdA2-CcdB2 heterohexamer, combined with residues 41–72 of the two CcdA peptides and the CcdB2 toxin from structure 3G7Z. The geometry of this minimal helical unit was then optimized using Coot (). The helical symmetry of the 3D reconstruction was applied to this minimal helical unit to obtain a continuous model consisting of alternating CcdA2 and CcdB2 dimers. For the thick helices, two additional strands were generated by translating the minimal helical unit by 67.2 Å and 134.4 Å along the helical axis, and again applying the helical symmetry. Model building was performed in UCSF Chimera (). […]

Pipeline specifications

Software tools SPARX, Coot, UCSF Chimera
Databases CCDB
Applications cryo-EM, Protein structure analysis