Dörner Katerina, Martin-Garcia Jose M, Kupitz Christopher, Gong Zhen, Mallet T Conn, Chen Liqing, Wachter Rebekka M, Fromme Petra
School of Molecular Sciences, Arizona State University, Box 871604, Tempe, Arizona 85287, United States.
Center for Membrane Proteins in Infectious Diseases (MPID), Arizona State University, Box 871604, Tempe, Arizona 85287, United States.
Cryst Growth Des. 2016 Jul 6;16(7):3838-3845. doi: 10.1021/acs.cgd.6b00384. Epub 2016 May 10.
A new approach is described to screen for protein nanocrystals based on the reversibility of crystallization. Methods to characterize nanocrystals are in strong need to facilitate sample preparation for serial femtosecond X-ray nanocrystallography (SFX). SFX enables protein structure determination by collecting X-ray diffraction from nano- and microcrystals using a free electron laser. This technique is especially valuable for challenging proteins as for example membrane proteins and is in general a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis. Nanocrystal growth cannot be monitored with common methods used in protein crystallography, as the resolution of bright field microscopy is not sufficient. A high-performance method to screen for nanocrystals is second order nonlinear imaging of chiral crystals (SONICC). However, the high cost prevents its use in every laboratory, and some protein nanocrystals may be "invisible" to SONICC. In this work using a crystallization robot and a common imaging system precipitation comprised of nanocrystals and precipitation caused by aggregated protein can be distinguished.
本文描述了一种基于结晶可逆性筛选蛋白质纳米晶体的新方法。为了便于进行串联飞秒X射线纳米晶体学（SFX）的样品制备，亟需表征纳米晶体的方法。SFX通过使用自由电子激光收集纳米晶体和微晶体的X射线衍射来确定蛋白质结构。该技术对于诸如膜蛋白等具有挑战性的蛋白质尤为有价值，并且总体上是克服辐射损伤问题和进行时间分辨结构分析的有力方法。由于明场显微镜的分辨率不足，蛋白质晶体学中常用的方法无法监测纳米晶体的生长。一种筛选纳米晶体的高性能方法是手性晶体的二阶非线性成像（SONICC）。然而，其高昂的成本使其无法在每个实验室使用，并且一些蛋白质纳米晶体可能对SONICC“不可见”。在这项工作中，使用结晶机器人和普通成像系统，可以区分由纳米晶体组成的沉淀和由聚集蛋白质引起的沉淀。
