Computational protocol: Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae

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Protocol publication

[…] SAXS data was measured at beamline BM29 at the ESRF using a X-ray wavelength of 1 Å at 10°C. Two samples of DsLOV with concentrations of 7.2 and 14.8 mg/ml were measured in phosphate buffer (10 mM NaH2PO4, 300 mM NaCl, pH 8.0). The buffer was measured before and after each protein sample. The samples were purged through a quartz capillary during X-ray exposure. For each sample ten frames with an exposure time of 2 sec each were recorded, and the frames without radiation damage were merged.Data analysis was done using the ATSAS software package []. Measured data were scaled by the concentration, where the lower concentration was used for the smaller q-range, while the data at higher concentration was taken for the high q-range. The excluded volume was calculated with the program DATPOROD and the molecular mass was estimated by applying a division factor of 1.7 []. Theoretical scattering curves of the crystallographic structures of the monomer and the dimer were calculated and fitted to the experimental SAXS data using the program CRYSOL. The program EOM was used to generate 10000 native-like conformations of N-terminal residues 1–19 and the C-terminal His6-tag (LEHHHHHH), while the crystal structure of the dimer was held fixed. Consecutively, representative ensembles were selected, which best describe the measured data. The distance distribution function P(r) was determined using the program DATGNOM. Ab initio models were generated using the programs GASBOR and DAMMIF applying two-fold symmetry. In both cases 20 ab initio models were generated, averaged and the filtered model was used. The molecular mass was estimated from the excluded volume of the filtered DAMMIF ab initio model using a division factor of 2 []. The crystal structure of the dimer was aligned in the filtered model. The envelope function was determined using the SITUS package. [...] Prior to cryo cooling, the crystals were soaked stepwise in reservoir solution containing up to 20% (v/v) glycerol. Both X-ray diffraction data were collected at 100 K, one under constant dark and one under constant light conditions. In the light condition, the crystals were exposed to blue-light prior to cryo cooling and in the subsequent steps. Please note that the crystals used for the datasets reported here were from the same drop and were stored in the dark at 100 K (in cryo tanks) until data collection. The X-ray diffraction data were recorded at beamline ID14-1 and ID23-1 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The data collection strategies taking radiation damage into account were based on calculations using the program BEST []. Data processing was carried out using MOSFLM and SCALA, which is part of the CCP4 software package []. The dark state structure was determined by molecular replacement using PHASER [part of the PHENIX software suite []]. The search model was built by homology modeling via the ExPASy web server [,], based on the template of the PDB ID 2V0U (phototropin 1 from Avena sativa). Phase determination was followed by several cycles of automated model building and refinement using the PHENIX package. The model was further improved by manual rebuilding using the program COOT []. The refinement and rebuilding of the photoexcited structure was carried out with the finished dark state structure. For statistics on data collection and refinement refer to Table .Unless otherwise indicated, figures were generated with MOLSCRIPT [] and RASTER3D [] using secondary structure assignments as given by the DSSP program []. […]

Pipeline specifications

Software tools ATSAS, CRYSOL, EOM, GASBOR, DAMMIF, CCP4, PHENIX, Coot, MolScript, Raster3D
Databases ExPASy
Applications Drug design, Small-angle scattering, Protein structure analysis
Organisms Dinoroseobacter shibae
Diseases Bacterial Infections
Chemicals Cysteine, Hydrogen, Oxygen, Sulfur