Computational protocol: Structural photoactivation of a full-length bacterial phytochrome

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Protocol publication

[…] The pair distance distribution functions of scattering curves was calculated using GNOM (). Difference scattering data were processed using our own code, and conventional SAXS parameters for all values of α were computed using programs from the ATSAS package (), with the exception of the SAXS invariant Vc and molecular mass that were calculated as described by Rambo and Tainer (). [...] To assess the overall structure of the Pr and Pfr states, ab initio models were generated using DAMMIN (). Models were calculated in rounds. Twenty DAMMIN models were calculated for each scattering curve. Because phytochromes are expected to be elongated homodimeric proteins, the symmetry was fixed as P2, and the shape was assumed to be prolate along the protein. The assumption on twofold symmetry rests on the solved crystal structures of the D. radiodurans phytochrome photosensory domain [for example, PDB code 4Q0J ()] as well as the near-symmetric arrangement seen in histidine kinases (). The starting shape was a sphere. For each curve, the 20 generated models were aligned using DAMAVER (). This gave a model that was used as input for a second DAMMIN round to generate 10 additional structures. The resulting models were compared using DAMSEL, and the model with least average normalized spatial discrepancy was chosen as the representative model. [...] A homology model for the full-length D. radiodurans phytochrome was constructed by combining known structures and homology information. The output domain was modeled using the SWISS-MODEL web interface (), with the cytoplasmic portion of a histidine kinase from T. maritima (PDB code 2C2A) as a monomeric template. The model dimer was then derived using symmetry operations of the template structure, and the histidine kinase model extended in the N-terminal direction with an ideal helix containing the D. radiodurans phytochrome residues 492 to 501.The model of the output domain was combined with our previously proposed solution structure of the Pr state of PAS-GAF-PHY () in a number of steps. First, to reconcile the registry mismatch between the two parts of the connecting helices from the PAS-GAF-PHY fragment and the model output domain, a series of molecular dynamics simulations were run, where the backbone of residues 492 to 501 on the output domain was restrained by progressively higher force constants to overlap with the corresponding residues on the PAS-GAF-PHY fragments. Next, after merging the models to form the full-length molecule, these restraints were progressively removed, and the new helical arrangement was found to be stable. Last, nonpolar contacts and salt bridges between the connecting helices of opposing monomers were manually formed in a short simulation where these residues (495-496, 496-495, 502-502, 509-509, and 516-516) were artificially bound together, after which they were stable in all subsequent runs.The quality of the final homology model, although subjected to the choices made during its construction, was evaluated by running a 500-ns equilibrium simulation, with all restraints removed. The region connecting the PAS-GAF-PHY fragment with the output domain was seen to be entirely stable, whereas the positions of the loosely linked CA domains were incompletely sampled on this time scale. […]

Pipeline specifications

Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Deinococcus radiodurans