Serial Para Crystal Reports 2011 Sp2
Serial Para Crystal Reports 2011 Sp2 ->>->>->> https://tlniurl.com/2tsZzC
We present here the first crystal structure of a Type IIL RM enzyme bound to its DNA substrate. MmeI differs from conventional Type II R-M systems (such as BamHI or EcoRI) in that the DNA recognition, methyltransferase, and endonuclease activities reside within the same polypeptide. The fact that the same DNA recognition module is responsible for host modification and endonuclease functions makes MmeI (and related enzymes) much more amenable to changes in DNA-binding and -cleavage specificities than conventional Type II enzymes. Based on bioinformatics analysis alone, we have rationally engineered dozens of MmeI-like enzymes with new specificities . These specificity changes are at positions 3, 4, and 6 of the MmeI recognition sequence (TCCRAC), and the engineered enzymes have specific activities that are comparable to the wild-type enzyme. The DNA-bound MmeI structure provides a molecular basis for these specificity changes and reveals new interactions to guide the engineering of additional enzymes.
The X-ray diffraction data on the MmeI/DNA/Sinefungin co-crystals were measured at the Advanced Photon Source at the Argonne National Laboratory. The data on native crystals were measured at beamline 23ID-D at a wavelength of 0.91938 Å, while single wavelength anomalous data on a Se-Met crystal were measured at a wavelength of 0.97944 Å (Se-K absorption edge) at the beamline 24ID-C. The HKL2000 package  was used to merge and scale X-ray data. Both the native and Se-Met crystals belong to space group P1. The unit-cell dimensions of native crystals are a = 61.87 Å, b = 95.29 Å, c = 161.96 Å, α = 72.84, β = 89.15, and γ = 71.61; and unit-cell dimensions of the Se-Met crystals are a = 62.08 Å, b = 94.68 Å, c = 159.91 Å, α = 73.34, β = 80.35, and γ = 71.89. The structure was solved using SAD phasing method using SHARP . The electron density map derived from experimental phasing was readily interpretable and showed clear electron density of both protein and DNA molecules. The model was built manually using program Coot  and iteratively refined with the program package Phenix  to the 2.6 Å resolution limit of the native crystals (Table 1). The final model contains two molecules of MmeI bound to two separate DNA duplexes and two Sinefungin moieties. The quality of the structure is excellent, with >97% of the residues in the most favored regions of the Ramachandran plot (Table 1). 1e1e36bf2d