Membrane Protein Structure by Electron Crystallography

描述(由申请人提供)：这项建议的总体目标是促进通过2D晶体的低温电子显微镜(Cryo-EM)来确定完整的膜蛋白的结构。膜蛋白对所有细胞都是必不可少的，也是最受欢迎的药物靶点，但其3D结构已被证明很难确定。Cryo-EM具有在原子分辨率下确定结构的能力，并具有在天然膜环境中保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激增给X射线结晶学带来了明显的优势。我们建议实施类似的技术来筛选脂双层内完整膜蛋白的2D结晶试验，以便在高通量的后基因组时代重新确立冷冻-EM作为可行的替代方案。我们将与纽约膜蛋白结构联盟(NYCOMPS)建立合作伙伴关系，NYCOMPS是NIH蛋白质结构倡议的一个专门中心，由纽约结构生物学中心运营。NYCOMPS将从他们正在筛选表达水平和同质性的膜蛋白大型数据库中提供所需的表达载体。在放大表达后，我们将使用机器人液体处理机通过透析建立2D结晶实验，并通过96孔格式的负染色制备EM样本。成像目前是筛选2D晶体试验的关键瓶颈，我们将实施从电子显微镜中的这些样品自动获取图像的技术。因此，我们预计每年评估50个不同的蛋白质目标。由此得到的信息将被用来建立管理2D结晶过程的一般原则，更重要的是，用于生产适合于以原子分辨率确定结构的2D晶体。给出了一种系统地寻找和优化2D晶体的可行方法，低温EM将成为高分辨率结构测定的普遍可行的方法，并提供一种重要的替代X射线结晶学和核磁共振光谱的方法。特别是，对蛋白质的数量和纯度的适度要求，以及脂膜提供的自然环境，使冷冻-EM成为一个有吸引力的替代方案，特别是当我们从细菌蛋白质组转向来自真核细胞的大型膜蛋白复合体时，事实证明，这些复合体更难表达和在洗涤剂溶解状态下保持。

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.