Biomedical Imaging

成像科学实验室参与了与美国国立卫生研究院的一项重大合作研究工作，涉及使用结构生物学中的图像处理技术和先进的计算技术来分析电子显微图像和核磁共振谱，目的是确定大分子结构和动力学。最近的工作集中在二十面体病毒衣壳结构的三维重建、分析和解释上，以及分离和复杂的蛋白质和核酸的结构确定和分析上。正在进行的研究包括分析与乳头瘤病毒和含有病毒衣壳的RNA有关的结构。此外，我们还一直在开发利用核磁共振数据研究生物大分子的结构和动力学的计算工具。我们开发和维护用于结构确定的Xplor-NIH软件包，该软件包在研究所的核磁共振实验室中使用，也在世界范围内使用。在过去的一年里，我们完成了用于核磁共振结构计算的隐式溶剂模型的开发，该模型允许比通常用于结构确定的原子-原子相互作用更真实。我们已经证明，使用这种新的方法可以提高计算结构的质量，并且可以在较少的实验数据存在的情况下进行准确的结构计算。在其他工作中，我们已经证明了HIV衣壳蛋白的结构可以在溶液中使用剩余偶极耦合(RDC)和小角X射线散射(SAXS)数据来确定。这种软性分子在正常的实验条件下形成单体和二聚体的混合物，这只能用结构系综来恰当地描述。这些结构的另一个复杂特征是单体系综不同于亚单位二聚体系综。 人类乳头瘤病毒(HPV)被认为是宫颈癌和其他上皮性癌症的病原体。主要衣壳蛋白(L1)最初形成一个松散连接的前衣壳，在体外条件下，它在几个小时内浓缩成更熟悉的直径60纳米的乳头瘤病毒衣壳。在这个过程中，原衣壳的直径缩小了5%；它的五聚体胶囊在结构上发生了变化，最明显的是在轴向区域；相邻胶囊之间的相互作用表面得到了巩固。这些结构变化伴随着二硫键交联物的形成，这些交联物增强了成熟衣壳的稳定性。将裂解物缓冲到更中性的条件下，可以生产具有90%二硫键的重组衣壳蛋白。生产完全成熟的重组衣壳的能力应该有利于对天然HPV衣壳进行进一步的结构研究。此外，完全成熟的假病毒粒子应被视为旨在阐明HPV在自然感染过程中使用的进入途径的研究的首选试剂。目前的工作包括合作，目标是设计出更好的HPV疫苗。HPV还含有少量衣壳蛋白(L2)。L1序列并不是在所有类型的HPV中都高度保守。另一方面，L2是高度保守的。目前(含L1)疫苗大约85%有效，因为它们含有HPV亚型的混合物。然而，由于L2高度保守，基于L2的疫苗可以提供接近100%的疗效。我们的目标是研究更好的候选疫苗的结构(即试图达到接近100%的效果)。这是一个高风险、高回报研究的例子。 另一个长期结构项目研究含有病毒的双链RNA。这项研究正在进行中。 蛋白质在溶液中具有多种生物重要构象。虽然核磁共振和小角散射数据(SAS)是很好的溶液结构探针，但同时确定多个结构及其种群是一个困难的问题，这是我们在过去15年中一直在解决的问题。我们最近应用这些方法来阐明磷酰化转移系统的酶I蛋白中的超大规模运动，这对 调节细胞对糖的吸收。 我们与加州大学圣地亚哥分校和Sanford Burnham Prebys医学发现研究所的研究人员合作，开发并应用了一种隐含溶剂模型，用于与实验数据一起用于确定溶液相和膜蛋白的结构 结构。通常，核磁共振结构计算是在没有溶剂的情况下进行的。在隐式模型中加入溶剂效应已被证明可以改善这些类型蛋白质的计算结构。 我们还回顾了用于RNA核磁共振结构确定的简化力场，并对多个方面进行了替换和优化，包括共价几何和原子半径参数，以及用于保持二面角的合理值的基于知识的平均力势的现代化。由此产生的力 FIELD在很大程度上有助于改进核磁共振RNA结构，使其在使用标准验证工具进行评估时接近X射线晶体结构的质量。 我们开发并推广了将电子显微镜(EM)数据与核磁共振数据结合在一起进行原子级结构确定的计算方法。这项工作已经出版了两本书。其中一个详细说明了EM数据在确定核磁共振结构中的作用。在第二篇论文中，核磁共振数据允许 确定与大离子通道蛋白结合的蛋白毒素配体的构象，该离子通道蛋白涉及热敏，这是以前使用EM确定的。

The Imaging Sciences Laboratory is involved in a major collaborative research effort with the NIH Institutes involving the use of image processing techniques and advanced computational techniques in structural biology to analyze electron micrographs and NMR spectra with the goal of determining macromolecular structures and dynamics. Recent efforts have concentrated on the 3D reconstruction, analysis and interpretation of the structures of icosahedral virus capsids in addition to structure determination and analysis of isolated and complexed proteins and nucleic acids. Ongoing research involves analyses of structures related to papillomavirus and RNA containing virus capsids. In addition we have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using Nuclear Magnetic Resonance (NMR) data. We develop and maintain the Xplor-NIH software package for structure determination, which is used in the NMR labs in the Institutes, and also worldwide. In the past year we have completed the development of an implicit solvent model to use with NMR structure calculations, which allows for much more realistic atom-atom interactions than are usually used in structure determination. We have shown that the use of this new approach can improve the quality of calculated structures, and can allow for accurate structure calculation in the presence of less experimental data. In other work, we have shown that the structure of the HIV capsid protein can be determined in solution using residual dipolar coupling (RDC) and small angle X-ray scattering (SAXS) data. This floppy molecule forms a mixture of monomer and dimer in normal experimental conditions, which can only properly be described using an ensemble of structures. A further complicating characteristic of these structures is that the monomer ensemble is different from the subunit dimer ensemble. Human papillomavirus (HPV) has been implicated as the causative agent in cervical and other epithelial cancers. The major capsid protein (L1), initially forms a loosely connected procapsid which, under in vitro conditions, condenses over several hours into the more familiar 60 nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter; its pentameric capsomeres change in structure, most markedly in their axial region; and the interaction surfaces between adjacent capsomeres are consolidated. These structural changes are accompanied by the formation of disulfide crosslinks that enhance the stability of the mature capsid. Buffering the lysate to more neutral conditions allowed the production of recombinant capsids with >90% disulfide bonds. The ability to produce fully mature recombinant capsids should benefit further, structural investigations of native HPV capsids. Moreover, fully mature pseudovirions should be viewed as preferred reagents for studies aimed at elucidating the entry pathways used by HPV in the course of natural infections. Current work involves collaborations with a goal to engineer a better HPV vaccine. HPV also contains a minor capsid protein (L2). The L1 sequence is not highly conserved across all types of HPV. L2, on the other hand, is highly conserved. Current (L1 containing) vaccines are roughly 85% effective because they contain a mixture of HPV subtypes. However, since L2 is highly conserved, a vaccine based on L2 could provide close to 100% efficacy. Our goal is to study the structure of a better vaccine candidates (i.e. to try to achieve closer to 100% effectiveness). This is an example of Hi-Risk, Hi-Reward research. Another long-term structural project studies double-stranded RNA containing viruses. This research is ongoing. Proteins take multiple, biologically important conformations in solution. While NMR and small angle scattering data (SAS) are good probes of solution structures, simultaneously determining multiple structures along with their populations is a difficult problem, one which we have been addressing over the past 15 years. We have recently applied these approaches to elucidate very large-scale motion in the Enzyme I protein of the phosphoryl transfer system, important for regulation of sugar uptake by cells. In collaboration with researchers at the University of California, San Diego and the Sanford Burnham Prebys Medical Discovery Institute, we have undertaken the development and application of an implicit solvent model for use in conjunction with experimental data in the structure determination of solution-phase and membrane protein structures. Normally, NMR structure calculations are carried out in the absence of solvent. Including solvent effects in an implicit model has been shown to improve the calculated structures of these types of proteins. We have also revisited the simplified force field used in NMR structure determination of RNA, and replaced and optimized multiple aspects, including parameters for covalent geometry and atomic radii, and modernizing a knowledge-based potential of mean force used to maintain reasonable values of the dihedral angles. The resulting force field greatly assists in improving NMR RNA structures such that they approach the quality of X-ray crystal structures when evaluated using standard validation tools. We have developed and promoted computational methods for incorporating electron microscopy (EM) data into atomic-level structure determination, in concert with NMR data. This work has resulted in two publications. One details the usefulness of EM data in NMR structure determination of NMR. In a second paper NMR data allowed the determination of the conformation of a protein toxin ligand bound to a large ion channel protein involved in heat sensing which had previously been determined using EM.