Computational protocol: Modulation of inhibitory activity of xylanase - α-amylase inhibitor protein (XAIP): binding studies and crystal structure determination of XAIP- II from Scadoxus multiflorus at 1.2 Å resolution

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Protocol publication

[…] The X-ray intensity data were collected using the DBT-sponsored synchrotron beamline ID 14-2 at the European Synchrotron Research Facility (ESRF) in Grenoble (France) using a MAR CCD detector (MAR USA Inc., Evanston, USA). In order to minimize the radiation damage, the crystal was placed in a nylone loop and kept at 100K in nitrogen stream during the measurements. The water ice formation was avoided by pre-incubation of the XAIP-II crystals for 3 minutes in the reservoir solution containing 22% (v/v) glycerol. The observed reflection data extended to a maximum resolution of 1.2Å. The reflection data were processed using DENZO and SCALEPACK from the HKL-2000 package []. Further data processing was carried out using programs from CCP4 package []. The crystals belong to monoclinic space group P21 with unit cell dimensions of a = 42.2 b = 64.3, c = 48.6Å and β = 102.1° with one molecule in the asymmetric unit of the crystal unit cell. The crystal packing parameter Vm was calculated to be 2.2Å3/Da which corresponded to a solvent content of 44%. The data collection and processing statistics are summarized in Table . [...] The structure of XAIP-II was determined with molecular replacement method using coordinate of XAIP (PDB: 3D5H) as the search model. It was refined using the options of rigid body refinement, simulated annealing and energy minimization with program CNS [] using data in the resolution range of 36.0 to 1.2Å. The conformations of loops were particularly examined by inspecting the composite OMIT maps using the programs REFMAC5 from CCP4 program suite [] and COOT []. The refinement steps were repeated with intermitant manual building of the model. The positions of water oxygen atoms were determined manually using difference Fourier (|Fo - Fc|) maps on the basis of peak height and distance criteria. The water molecules whose thermal factors were 50Å2 or above after refinement were removed from the list. Further model building and refinement cycles resulted in an Rcryst of 0.158 and Rfree of 0.186 for 62,459 reflections from 36.0 to 1.2Å resolution. The average value of thermal B factor for all the atoms was 15.8Å2. The refinement statistics is given in Table . […]

Pipeline specifications

Software tools HKL-2000, CCP4, CNS, REFMAC5, Coot
Application Protein structure analysis