Shi Dan, Nannenga Brent L, Iadanza Matthew G, Gonen Tamir
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
Elife. 2013 Nov 19;2:e01345. doi: 10.7554/eLife.01345.
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1-1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name 'MicroED', that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001.
我们证明，通过在电子冷冻显微镜（CryoEM）中对三维晶体进行电子晶体学来确定高分辨率蛋白质结构是可行的。溶菌酶微晶被冷冻在电子显微镜网格上，并收集了分辨率达1.7 Å的电子衍射数据。我们开发了一种数据收集方案，以收集电子衍射中的全倾斜系列数据直至原子分辨率。单个倾斜系列包含从单个晶体收集的多达90个单独的衍射图案，倾斜角增量为0.1 - 1°，且总累积电子剂量小于每埃平方10个电子。我们对来自三个晶体的数据进行了索引，并通过分子置换随后进行晶体学精修至2.9 Å分辨率，将这些数据用于溶菌酶的结构测定。这一原理验证为一种我们命名为“MicroED”的新技术的实施铺平了道路，该技术可能在结构生物学中具有广泛的适用性。DOI: http://dx.doi.org/10.7554/eLife.01345.001 。
