Technology Development for 3D Electron Microscopy

3D电子显微镜技术开发

• 批准号: 8937860
• 负责人: Sriram Subramaniam
• 金额: $ 81.49万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937860
• 关键词: AIDS/HIV problem; Address; Antibodies; Area; Binding; Biological; Biology; Cancer Biology; Charge; Classification; Complex; Cryoelectron Microscopy; Crystallization; Development; Dimensions; Dose; Electron Beam; Electron Microscopy; Electrons; Enzymes; Exposure to; Financial compensation; Foundations; HIV; Hybrids; Image; Imaging technology; Individual; Infection Control; Influenza; Influenza A Virus, H1N1 Subtype; Ion Channel; Joints; Laboratories; Length; Location; Malignant Neoplasms; Maps; Measures; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Motion; NMR Spectroscopy; Noise; Predisposition; Procedures; Process; Proteins; Radiation; Relative (related person); Reporting; Resolution; Ribosomes; Side; Signal Transduction; Staging; Structural Models; Structure; Surface; Suspension substance; Suspensions; Techniques; Three-Dimensional Image; Time; Vaccine Design; Virion; Virus; Work; X-Ray Crystallography; base; beta-Galactosidase; density; detector; electron tomography; frontier; improved; influenza virus vaccine; metalloenzyme; molecular imaging; movie; multicatalytic endopeptidase complex; neutralizing antibody; new technology; pandemic disease; particle; protein complex; protein structure; reconstruction; stem; structural biology; technology development; three dimensional structure; tool; two-dimensional; virology

A number of new technologies have been developed from our work over the last few years that are now at the core of our discovery efforts in the areas of HIV/AIDS and cancer biology. Two advances are especially important for our work at the intersection of structural biology and virology. We extended our efforts in subvolume averaging of cryo-electron tomographic volumes by applying automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. One useful recent biological application of our methods has been in the area of vaccine design in influenza to determine the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. To address this, we first obtained 3D structures from cryo-electron tomography of HA on intact 2009 H1N1 pandemic virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes including H1, H2, H5, H6 and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the relative locations of the molecular surfaces of HA involved in interaction with C179. To separate any unliganded from antibody-bound HA spikes, we used a newly developed procedure for collaborative alignment. Our approach uses an iterative Joint Alignment-Classification (JAC) technique, in which the classical similarity measure based on pair-wise distances between two particles, or a particle and a class average, is replaced by a one-to-many collaborative similarity function measured between a particle and a group of particles. This collaborative alignment framework enables robust alignment of linearly correlated heterogeneous images. Our analysis shows that despite their close packing on the surface of the virus, 75% of all HA trimers are complexed with the antibody, establishing that universal influenza vaccines that elicit such antibodies could be effective in controlling the infection. In a different development, we have made significant inroads to develop the potential of obtaining near-atomic resolution structures using cryo-EM. Atomic resolution models for proteins and protein complexes are usually obtained using X-ray crystallography or NMR spectroscopy, and in selected instances, by cryo-EM of ordered protein assemblies. The vast majority of high resolution structures obtained using cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or two-dimensional crystals. We have now shown that emerging methods in single-particle cryo-EM can be used for structure determination at near-atomic resolution, even for much smaller protein complexes with low symmetry, by determining the structure of the 465-kDa enzyme beta-galactosidase. Our 3.2 Angstrom structure for beta-galactosidase represents the highest resolution achieved so far by single particle cryo-EM for a protein complex smaller than 500 kDa. In recent developments, structures of a ribosome complex (more than 2000 kDa), metalloenzyme complex (1200 kDa), proteasome (700 kDa) and an ion channel (300 kDa) have been reported at resolutions of 3.2, 3.4, 3.3 and 3.4 Angstroms, respectively. The advances that we and others have now made in determination of structures of protein complexes and membrane proteins at near-atomic resolution mark a critical shift in structural biology. One reason for the excitement is the demonstrated prospect that at least in some instances, it may be possible to dispense with the complicated process of crystallizing proteins in order to determine their structure. Another reason is the fact that atomic resolution structures can be determined from just a few microliters of a protein suspension, and from as few as 12,000 molecular images. There are other technical "firsts" in our work. We present evidence for the first time for the selective susceptibility of negatively charged residues to radiation damage that occurs with exposure to electron beams. We show that fractionating the electron dose allows one to maximize the level of structural detail that is visualized in density maps obtained by cryo-electron microscopy. Thus, using only the first few exposures enabled determination of maps where the densities of all side chains could be visualized clearly, before the selective degradation of density from negatively charged residues. Further, our studies show that atomic resolution structures can be obtained even from regions in proteins that are normally altered or deleted to obtain diffracting crystals, and provide a new dimension for the use of hybrid approaches for structure-function studies of intact, full-length protein complexes. Two other advances that helped achieve high resolution were improvements in methods for compensation of drift and beam induced motion during the electron exposure, and in more accurate determination of contrast transfer function (CTF) parameters, as discussed below. For drift correction, the cumulative averages of previously aligned frames were used as a reference to align individual frames of each movie by cross-correlation. The increased signal-to-noise ratio of the cumulative average of frames which was used as a reference to align the raw frames, results in significant improvements in the accuracy of motion correction as evidenced by better defined Thon rings in the drift-corrected images. Accuracy of CTF determination is one of the main factors that impact resolution in single-particle cryo-EM especially in the near-atomic resolution regime. Taking advantage of the capability of direct electron detectors to produce dose fractionated movies, we show that in addition to providing opportunities for motion correction and mitigation of radiation damage effects, movies also provide a way to improve the accuracy of CTF determination and therefore the resolution of single-particle reconstructions. The way we accomplish this is by estimating the parameters of the CTF of each micrograph using radially averaged power spectra obtained by periodogram averaging with tiles extracted from all frames of each movie. Compared to the standard approach of using tiles extracted from the average of frames (after drift-correction), our method does not require frames to be pre-aligned and results in better-defined 1D CTF profiles allowing more accurate determination of defocus. The improvement in the resolvability of zero-crossings obtained using this approach allowed us to set the upper resolution limit for CTF estimation at 3 Angstromwhile still providing reliable CTF fits. The prospect that the determination of protein structures to atomic resolution will no longer be limited by size or by the need for crystallization represents a significant and exciting horizon in structural biology. Rather than simply using cryo-EM maps, typically in the 6 - 20 Angstrom resolution range, as an envelope in which to fit structures obtained by X-ray crystallography, there is the exciting prospect of using cryo-EM to derive de novo, high-resolution structural models of proteins in one or multiple functional conformational states. The stage is now set for the application of these methods to analyze structures of a wide variety of biologically and medically relevant multi-protein complexes and membrane protein assemblies, which have historically represented the most challenging frontier in structural biology.

我们在过去几年的工作中开发了许多新技术，这些技术现在是我们在艾滋病毒/艾滋病和癌症生物学领域发现工作的核心。在结构生物学和病毒学的交叉领域，有两项进展对我们的工作尤其重要。我们通过应用自动化，迭代，缺失楔形校正的3D图像对齐和分类方法来区分同时存在的多种构象，扩展了我们在低温电子层析体积亚体积平均方面的努力。我们的方法最近的一个有用的生物学应用是在流感疫苗设计领域，以确定病毒表面的血凝素干区在多大程度上可以接触到广泛中和的抗体。为了解决这个问题，我们首先通过冷冻电子断层扫描获得了在存在和不存在抗体C179的情况下，完整的2009 H1N1大流行病毒粒子上的HA的三维结构，C179可以中和表达多种HA亚型的病毒，包括H1， H2, H5， H6和H9。通过将先前得到的三聚体HA的晶体结构拟合到密度图中，我们推断出与C179相互作用的HA分子表面的相对位置。为了从抗体结合的HA尖峰中分离任何非配体，我们使用了一种新开发的协作比对程序。我们的方法使用迭代联合对齐-分类（JAC）技术，其中基于两个粒子之间的成对距离或粒子与类平均值的经典相似性度量被一个粒子与一组粒子之间测量的一对多协作相似性函数所取代。这种协作对齐框架可以实现线性相关异构图像的鲁棒对齐。我们的分析表明，尽管HA三聚体在病毒表面紧密包裹，但75%的HA三聚体与抗体络合，这表明引发此类抗体的通用流感疫苗可能有效控制感染。在另一个不同的发展中，我们已经取得了重大进展，以开发利用低温电镜获得近原子分辨率结构的潜力。蛋白质和蛋白质复合物的原子分辨率模型通常使用x射线晶体学或核磁共振光谱获得，在某些情况下，通过有序蛋白质组装的冷冻电镜获得。绝大多数使用低温电镜获得的高分辨率结构通常局限于大的、有序的实体，如螺旋或二十面体组装或二维晶体。通过测定465 kda酶-半乳糖苷酶的结构，我们已经证明单粒子低温电镜中的新兴方法可以用于近原子分辨率的结构测定，甚至可以用于低对称性的小得多的蛋白质复合物。我们的β -半乳糖苷酶的3.2埃结构代表了迄今为止单颗粒低温电镜对小于500 kDa的蛋白质复合物所取得的最高分辨率。在最近的发展中，核糖体复合物（超过2000 kDa）、金属酶复合物（1200 kDa）、蛋白酶体（700 kDa）和离子通道（300 kDa）的结构分别在3.2、3.4、3.3和3.4埃的分辨率上被报道。我们和其他人在近原子分辨率下测定蛋白质复合物和膜蛋白结构方面取得的进展标志着结构生物学的一个关键转变。令人兴奋的一个原因是，至少在某些情况下，为了确定蛋白质的结构，有可能省去使蛋白质结晶的复杂过程。另一个原因是，原子分辨率结构可以从几微升的蛋白质悬浮液中确定，也可以从12000个分子图像中确定。在我们的工作中还有其他技术上的“第一”。我们首次提出了带负电荷的残基对暴露于电子束下的辐射损伤的选择性敏感性的证据。我们表明，分离电子剂量可以最大限度地提高结构细节的水平，这是通过低温电子显微镜获得的密度图可视化。因此，在负电荷残基选择性降解密度之前，仅使用前几次暴露就可以确定所有侧链的密度可以清晰地可视化的地图。此外，我们的研究表明，原子分辨率结构甚至可以从蛋白质中通常被改变或删除以获得衍射晶体的区域获得，并为使用混合方法进行完整全长蛋白质复合物的结构-功能研究提供了新的维度。另外两个有助于实现高分辨率的进步是电子暴露过程中漂移和光束诱导运动补偿方法的改进，以及对比度传递函数（CTF）参数的更精确测定，如下所述。对于漂移校正，使用先前对齐帧的累积平均值作为参考，通过相互关联来对齐每个电影的单个帧。增加的累计平均帧的信噪比被用作对齐原始帧的参考，结果显著提高了运动校正的精度，这一点可以通过更好地定义漂移校正图像中的Thon环来证明。CTF测定的准确性是影响单粒子低温电镜分辨率的主要因素之一，特别是在近原子分辨率体系中。利用直接电子探测器产生剂量分割电影的能力，我们表明，除了提供运动校正和减轻辐射损伤效应的机会外，电影还提供了一种提高CTF测定精度的方法，从而提高了单粒子重建的分辨率。我们实现这一目标的方法是通过使用从每个电影的所有帧提取的瓦片进行周期图平均得到的径向平均功率谱来估计每个显微照片的CTF参数。与使用从帧的平均值中提取的瓦片（经过漂移校正）的标准方法相比，我们的方法不需要对帧进行预对齐，并且可以产生更好定义的1D CTF轮廓，从而更准确地确定离焦。使用这种方法获得的零交叉分辨率的提高使我们能够将CTF估计的分辨率上限设置为3埃，同时仍然提供可靠的CTF拟合。蛋白质结构的原子分辨率测定将不再受大小或结晶需要的限制，这一前景代表了结构生物学中一个重要而令人兴奋的前景。而不是简单地使用低温电镜图，通常在6 - 20埃的分辨率范围内，作为一个包络来适应x射线晶体学获得的结构，使用低温电镜来获得一个或多个功能构象状态的蛋白质从头开始，高分辨率的结构模型是令人兴奋的前景。现在，应用这些方法分析各种生物和医学相关的多蛋白复合物和膜蛋白组装体的结构的阶段已经确定，这历来是结构生物学中最具挑战性的前沿。