Biomedical Imaging

成像科学实验室参与了与NIH研究所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镜和NMR光谱，以确定大分子结构和动力学。最近的努力集中在三维重建，分析和解释的二十面体病毒衣壳的结构，除了结构测定和分析的分离和复合的蛋白质和核酸。正在进行的研究涉及与乳头瘤病毒和含有病毒衣壳的RNA相关的结构分析。此外，我们还一直在开发计算工具，用于使用核磁共振（NMR）数据研究生物大分子的结构和动力学。我们开发和维护用于结构测定的Xplor-NIH软件包，该软件包用于研究所的NMR实验室，也用于世界各地。在过去的一年中，我们已经完成了一个隐式溶剂模型的开发，用于NMR结构计算，它允许更现实的原子-原子相互作用比通常用于结构测定。我们已经表明，使用这种新的方法可以提高计算结构的质量，并可以允许在较少的实验数据的存在下进行准确的结构计算。在其他工作中，我们已经表明，HIV衣壳蛋白的结构，可以确定在溶液中使用残留偶极耦合（RDC）和小角X射线散射（SAXS）数据。这种松软的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描述。这些结构的另一个复杂特征是单体系综不同于亚基二聚体系综。 人乳头瘤病毒（HPV）是宫颈癌和其他上皮癌的致病因子。主要衣壳蛋白（L1），最初形成一个松散连接的原衣壳，在体外条件下，经过几个小时浓缩成更熟悉的60 nm直径的乳头状瘤病毒衣壳。在这个过程中，原衣壳的直径缩小了5%;它的五聚体壳粒结构发生了变化，最明显的是在它们的轴向区域;相邻壳粒之间的相互作用表面被巩固。这些结构变化伴随着二硫键交联的形成，其增强成熟衣壳的稳定性。将裂解物缓冲至更中性的条件允许产生具有>90%二硫键的重组衣壳。产生完全成熟的重组衣壳的能力应该有利于进一步的天然HPV衣壳的结构研究。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优选试剂。目前的工作涉及合作，目标是设计更好的HPV疫苗。HPV还含有次要衣壳蛋白（L2）。L1序列在所有类型的HPV中并不高度保守。L2是高度保守的。目前（含L1）疫苗的有效性约为85%，因为它们含有HPV亚型的混合物。然而，由于L2是高度保守的，基于L2的疫苗可以提供接近100%的功效。我们的目标是研究更好的候选疫苗的结构（即尝试实现接近100%的有效性）。这是高风险高回报研究的一个例子。 另一个长期的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学上重要的构象。虽然NMR和小角散射数据（SAS）是很好的探针的解决方案的结构，同时确定多个结构沿着与他们的人口是一个困难的问题，我们一直在解决在过去的15年。我们最近应用这些方法来阐明磷酰基转移系统中酶I蛋白的大尺度运动，这对于 调节细胞对糖的摄取。 我们与圣地亚哥的加州大学和Sanford Burnham Prebys医学发现研究所的研究人员合作，开发和应用了一种隐式溶剂模型，该模型与实验数据结合使用，用于确定溶液相和膜蛋白的结构 结构.通常，NMR结构计算在不存在溶剂的情况下进行。在隐式模型中包括溶剂效应已被证明可以改善这些类型蛋白质的计算结构。 我们还重新审视了用于RNA NMR结构测定的简化力场，并替换和优化了多个方面，包括共价几何形状和原子半径的参数，并使用于保持二面角合理值的平均力的基于知识的潜力现代化。的合力 场极大地帮助改进NMR RNA结构，使得它们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。 我们已经开发并推广了将电子显微镜（EM）数据与NMR数据相结合进行原子级结构测定的计算方法。这项工作已出版了两份出版物。一个详细的有用性的EM数据在NMR结构测定NMR。在第二篇论文中，NMR数据允许 确定与参与热感测的大离子通道蛋白结合的蛋白毒素配体的构象，所述热感测先前已使用EM确定。

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.