Membrane Protein Structure by Electron Crystallography

描述（由申请人提供）：本提案的总体目标是通过二维晶体的冷冻电子显微镜（cryo-EM）促进整体膜蛋白的结构测定。膜蛋白是所有细胞和药物靶点所必需的，但其三维结构已被证明难以确定。Cryo-EM具有在原子分辨率下确定结构的能力，以及在其天然膜环境中维持完整膜蛋白的重要优势。然而，用于3D结晶试验的高通量筛选的扩散给x射线晶体学带来了明显的优势。我们建议实施类似的技术来筛选脂质双层内整体膜蛋白的二维结晶试验，以便在高通量、后基因组时代重建冷冻电镜作为可行的替代方案。我们将与纽约膜蛋白结构协会（NYCOMPS）建立合作关系，该协会是美国国立卫生研究院蛋白质结构倡议的一个专业中心，在纽约结构生物学中心之外运作。NYCOMPS将从大型膜蛋白数据库中提供所需的表达载体，他们正在筛选表达水平和均匀性。在扩大表达后，我们将使用机器人液体处理器通过透析建立2D结晶试验，并通过96孔格式的阴性染色制备EM样品。成像目前是筛选二维晶体试验的关键瓶颈，我们将实现在电子显微镜下从这些样品中自动获取图像的技术。因此，我们预计每年评估-50个不同的蛋白质靶点。所得信息将用于建立控制二维结晶过程的一般原理，重要的是，用于生产适合原子分辨率结构测定的二维晶体。如果有一种可行的方法来系统地搜索和优化二维晶体，冷冻电镜将成为高分辨率结构测定的普遍可行方法，并提供x射线晶体学和核磁共振波谱学的重要替代方法。特别是，对蛋白质的数量和纯度的适度要求，以及脂质膜提供的自然环境，使冷冻电镜成为一个有吸引力的选择，特别是当我们从细菌蛋白质组转向真核细胞的大膜蛋白复合物时，这种复合物已被证明更难以表达和维持在洗涤剂溶解状态。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to promote structure determination of integral membrane proteins by cryoelectron microscopy (cryo-EM) of 2D crystals. Membrane proteins are essential to all cells and favored drug targets, yet their 3D structures have proven difficult to ascertain. Cryo-EM has a demonstrated capability for structure determination at atomic resolution and the important advantage of maintaining integral membrane proteins within their native membrane environment. Nevertheless, the proliferation of high- throughput screening for 3D crystallization trials has given a distinct advantage to X-ray crystallography. We propose to implement analogous technologies for screening 2D crystallization trials of integral membrane proteins within lipid bilayers in order to reestablish cryo-EM as a viable alternative in the high-throughput, post-genomic era. We will form a partnership with the New York Consortium on Membrane Protein Structure (NYCOMPS), which is a Specialized Center of the NIH Protein Structure Initiative operating out of the New York Structural Biology Center. NYCOMPS will provide requested expression vectors from a large database of membrane proteins that they are screening for expression levels and homogeneity. After scaling up expression, we will use a robotic liquid handler to set up 2D crystallization trials by dialysis and prepare EM samples by negative stain in a 96-well format. Imaging currently represents a critical bottleneck for screening 2D crystal trials and we will implement technologies to automatically acquire images from these samples in the electron microscope. Thus, we expect to assess -50 different protein targets per year. The resulting information will be used to establish general principles governing the 2D crystallization process and, importantly, to produce 2D crystals that are suitable for structure determination at atomic resolution. Given a workable method to systematically search for and optimize 2D crystals, cryo-EM will become generally viable for high resolution structure determination and offer an important alternative to X-ray crystallography and NMR spectroscopy. In particular, the modest requirements for quantity and purity of proteins, as well as the natural environment provided by the lipid membrane, make cryo-EM an attractive alternative, especially as we move from bacterial proteomes towards large membrane protein complexes from eukaryotic cells that have proven more difficult to express and to maintain in a detergent solubilized state.