Department of Structural Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Acta Crystallogr D Struct Biol. 2020 Dec 1;76(Pt 12):1222-1232. doi: 10.1107/S2059798320013741. Epub 2020 Nov 19.
The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.
衍射质量晶体的生长和实验相分析仍然是蛋白质晶体学的两个主要瓶颈。在这里，高亲和力的铜（II）结合三肽 GHK 被融合到 GFP 变体和 MBP-FG 肽融合物的 N 端。GHK 标签促进了结晶，来自对称分子的各种残基（His、Asp、His/Pro）完成了铜（II）的正方形-金字塔配位。通过铜单波长色散（SAD）相位测定可以快速确定结构，即使在非常低的 Bijvoet 比或经历显著辐射损伤后也是如此。当在接近铜吸收边缘的波长下收集高度冗余的数据时，残留的 S 原子位置也可以在对数似然梯度图中定位，并用于改善相位。GHK 铜单波长色散（SAD）方法为大分子结构的结晶和相分析提供了一种便捷的方法，并将补充当前向天然硫 SAD 和微晶体旋转（MR）SAD 相分析的趋势。
