Computational protocol: The processing of repetitive extragenic palindromes: the structure of a repetitive extragenic palindrome bound to its associated nuclease

Similar protocols

Protocol publication

[…] TnpAREP-REP crystals were cryoprotected using 80% mother liquor/20% glycerol mixture and flash cooled in liquid nitrogen. All diffraction data were collected at 95 K using Cu Kα radiation from a rotating anode source with multilayer focusing optics and an RAXIS IV image plate. The data were integrated and scaled with XDS (). The structure was determined by single-wavelength anomalous diffraction from two Hg atoms. SHELXD was used to locate them (), and their parameters were refined with SHARP (). The map was solvent flattened with Solomon (), and the model was built interactively with the program O (,). The structure was refined using CNS () with Cartesian simulated annealing, energy minimization and individual B factor refinement. The final model was refined with Refmac5, using restrained refinement (). Refinement was monitored by calculating Rfree using 5% of the data set aside for crossvalidation (). The final model was refined to an R of 22% and an Rfree of 28%. Drawings were prepared with PyMOL, ESPript and Adobe Illustrator (,). Additional X-ray data sets were collected on a single TnpAREP-REP crystal above the K absorption edge of Fe2+ (7200 eV) and Mn2+ (6700 eV), and below Mn2+ (6300 eV). Using another crystal two more data sets were collected at 8200 eV and at 8500 eV, which are below and above the K edge of Ni2+. These data were collected at the SERCAT beamline ID22 of the APS at the Argonne National Laboratory on a MAR300 CCD detector. […]

Pipeline specifications

Software tools SHELX, XDS, CNS, REFMAC5, PyMOL, ESPript, Adobe Illustrator
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Escherichia coli str. K-12 substr. MG1655
Chemicals Manganese, Nucleotides