Computational protocol: Crystal structure of a Pseudomonas malonate decarboxylase holoenzyme hetero-tetramer

Similar protocols

Protocol publication

[…] For the MdcD–MdcE complex, a single-wavelength anomalous diffraction dataset at 1.8 Å resolution was collected at 100 K at the peak absorption wavelength of selenium (0.9790 Å) at the X25 beamline of the National Synchrotron Light Source, using a Pilatus 6M detector. The diffraction images were processed using the HKL2000 package. The structure was solved using the program PHENIX. Manual model rebuilding was performed using Coot, and the structure refinement was carried out using PHENIX.The structure of the MDC hetero-tetramer was determined by the single isomorphous replacement method. A native dataset was collected at 3.0 Å resolution at the NE-CAT 24-IDC beamline at the Advanced Photon Source. A single-wavelength anomalous diffraction dataset at 3.5 Å resolution was collected at the peak absorption wavelength of gold (1.0402 Å) on the crystal soaked with K3Au(CN)6. Four gold sites were located with isomorphous difference Patterson map, as well as isomorphous difference electron density map using phases calculated from the MdcD–MdcE model, which was placed into the holoenzyme crystal by the molecular replacement method using the program COMO. The reflections were phased using the program PHENIX. Phase improvement, with two-fold non-crystallographic symmetry averaging, was carried out with Resolve. The models for MdcA and MdcC were built manually with Coot. The gold compound is bound to Cys22 of MdcC and Cys230 of MdcE, respectively.A new native crystal of the hetero-tetramer was produced in the presence of 20 mM 18-crown-6 crown ether as an additive, which diffracted to 2.2 Å resolution. The structure was solved by the molecular replacement method with the program COMO, and structure refinement was carried out with CNS and PHENIX. No electron density for the crown ether was observed in the crystal.The diffraction data on the CoA complex was processed with the program XDS. The structure of the complex was determined by the molecular replacement method with the program COMO, using the structure of one MDC hetero-tetramer as the search model. NCS restraints among the hetero-tetramers were used throughout the refinement. […]

Pipeline specifications

Software tools PHENIX, Coot, CNS, XDS
Applications Small-angle scattering, Protein structure analysis
Organisms Pseudomonas aeruginosa
Chemicals Biotin, Carbon, Coenzyme A