5v0q

Original engineered variant of I-OnuI meganuclease targeting the HIV integrase gene; harbors 49 point mutations relative to wild-type I-OnuI

spinqualitylabelsall models 5v0q, resolution 2.40Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5v0q is a 3 chain structure with sequence from Synthetic construct. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.4Å Ligands: CA Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner. Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.,Niyonzima N, Lambert AR, Werther R, De Silva Feelixge H, Roychoudhury P, Greninger AL, Stone D, Stoddard BL, Jerome KR Protein Eng Des Sel. 2017 Jul 1;30(7):503-522. doi: 10.1093/protein/gzx037. PMID:28873986 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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