Computational protocol: Combined SAXS/EM Based Models of the S. elongatus Post-Translational Circadian Oscillator and its Interactions with the Output His-Kinase SasA

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Protocol publication

[…] Crystals of the KaiC-ee mutant protein were grown under conditions similar to those established earlier for wt-KaiC from S. elongatus . Following mounting in a nylon loop and cryo-protection in 25% glycerol, diffraction data were collected on the 21-ID-F beamline of the Life Sciences Collaborative Access Team at the Advanced Photon Source (APS; Argonne National Laboratory, Argonne, IL) using a wavelength of 1.000 Å. Data were integrated and scaled with HKL2000 and the initial orientation of KaiC was established by molecular replacement with the program CNS , using as the search model the wt-structure (PDB ID 3DVL minus water and Mg2+ and with residues pS431 and pT432 mutated to alanine). Rigid body refinement was followed by cycles of positional refinement with CNS. Manual rebuilding was performed with the programs TURBO and COOT . Side chains of residues 431 and 432 were added and their conformations gradually adjusted in the six subunits, followed by further rounds of positional and isotropic B-factor refinement and addition of solvent molecules. Selected crystal data, data collection and refinement parameters are summarized in . Illustrations were generated with the program CHIMERA . [...] All small angle X-ray scattering data were collected on the SAXS/WAXS setup located at the 5-ID-D beamline of the DND-CAT synchrotron research center, Advanced Photon Source, Argonne National Laboratory (Argonne, IL). 1.2398 Å radiation was selected from the APS Undulator A spectrum using a Si-111 monochromator, with harmonic rejection provided by a 1∶1 horizontally focusing mirror, and collimated to 0.3×0.3 mm with a series of polished slits. The SAXS detector was a Mar-USA 162 mm CCD and covered the momentum transfer range 0.005GNOM package . [...] Molecular envelopes were calculated with the programs DAMMIN and GASBOR along with the averaging package DAMAVER , , using the SIBYLS SAXS ATSAS program ( ). Twenty averaged ab initio models were calculated and scored before selecting the most typical one. No symmetry restraints were applied in any of the shape reconstructions. The resulting NSD values () revealed a good similarity in shape for models of Kai proteins alone and the binary KaiC complexes, representative examples of which are depicted in the various figures. Three-dimensional structures of full-length proteins based on crystal structures (KaiA, KaiB and KaiC) or domains based on solution NMR (SasA) were built into the SAXS envelopes manually and optimized via rigid body refinement in some cases using CHIMERA . […]

Pipeline specifications

Software tools CNS, Coot, ATSAS, DAMMIN, GASBOR
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Synechococcus elongatus PCC 6301, Synechococcus elongatus
Chemicals Adenosine Triphosphate