Zhang Lei, Ren Gang
The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, USA.
J Phys Chem Biophys. 2012;2(2). doi: 10.4172/2161-0398.1000e103. Epub 2012 May 22.
The protein is naturally dynamic and heterogeneous in solution. Protein dynamics involves both equilibrium fluctuations that regulate biological function and other non-equilibrium effects of biological motors, which convert chemical energy to mechanical energy. However, a single, unique structure of protein determined from X-ray crystal and conventional single-particle electron microscopy is insufficient to encompass the dynamic nature of proteins in solution. Structure determination of dynamic and heterogeneous protein is essentially required the determination of each individual particle of protein. Recently, Drs. Gang Ren and Lei Zhan published the first single molecule three-dimensional (3D) EM images of individual proteins ever obtained with enough clarity to determine their structure, an IgG antibody (14 Å resolution) and a 17nm HDL (36 Å resolution). These results depended upon four innovations: i) improved cryo-electron microscopy (cryoEM) sample preparation and Electron microscopy (EM) operation conditions resulted in the successful imaging of a 17 nm HDL particle (120-200kDa) by cryo-electron tomography (cryoET); ii) developed an optimized NS (OpNS) protocol that eliminates the rouleau artifact that has plagued EM research for three decades. This OpNS protocol provides high-contrast single lipoprotein images with similar size (<5%) and shape (<5%) to that seen by cryoEM; iii) developed a high-resolution and high contrast sample preparation protocol, cryo-positive-staining (cryoPS) that allows direct visualization of the secondary structure of a small protein, such as the β-strands in CETP and the helical double belt of apoA-I in spherical HDL; iv) developed a robust tomography reconstruction method, Individual Particle Electron Tomography (IPET) that is a high-resolution, high throughput reconstruction method that, to the best of our knowledge, is the only method for determining an individual protein structure. Remarkably, IPET went against the conventional wisdom that a single protein can NOT be reconstructed by EM and this opens a door for the study of protein dynamics via a particle-by-particle structural comparison.
该蛋白质在溶液中天然具有动态性和异质性。蛋白质动力学既涉及调节生物功能的平衡波动，也涉及生物马达将化学能转化为机械能的其他非平衡效应。然而，通过X射线晶体学和传统单颗粒电子显微镜确定的单一、独特的蛋白质结构不足以涵盖溶液中蛋白质的动态性质。确定动态和异质蛋白质的结构本质上需要确定蛋白质的每个单独颗粒。最近，任刚博士和战雷博士发表了有史以来获得的单个蛋白质的首张单分子三维（3D）电子显微镜图像，清晰度足以确定其结构，一种IgG抗体（分辨率为14 Å）和一个17nm的高密度脂蛋白（HDL，分辨率为36 Å）。这些结果依赖于四项创新：i）改进的冷冻电子显微镜（cryoEM）样品制备和电子显微镜（EM）操作条件通过冷冻电子断层扫描（cryoET）成功成像了一个17nm的HDL颗粒（120 - 200kDa）；ii）开发了一种优化的NS（OpNS）方案，消除了困扰EM研究三十年的叠连伪影。这种OpNS方案提供了与cryoEM所见相似大小（<5%）和形状（<5%）的高对比度单脂蛋白图像；iii）开发了一种高分辨率和高对比度的样品制备方案，冷冻正染色（cryoPS），可直接观察小蛋白质的二级结构，如胆固醇酯转运蛋白（CETP）中的β链和球形HDL中载脂蛋白A-I的螺旋双带；iv）开发了一种强大的断层扫描重建方法，单颗粒电子断层扫描（IPET），这是一种高分辨率、高通量的重建方法，据我们所知，是确定单个蛋白质结构的唯一方法。值得注意的是，IPET违背了单颗粒蛋白质不能通过EM重建的传统观念，这为通过逐个颗粒的结构比较研究蛋白质动力学打开了一扇门。
