Biomedical Imaging

成像科学实验室与各研究所开展了一项重大的合作研究工作，涉及在结构生物学中使用图像处理技术和先进的计算技术来分析电子显微镜和核磁共振光谱，目的是确定大分子结构和动力学。近年来的研究主要集中在二十面体病毒衣壳的三维重建、分析和解释上。正在进行的研究涉及与疱疹病毒和乳头瘤病毒以及其他二十面体病毒衣壳相关的结构分析。 乳头瘤病毒（例如HPV-16）编码两种衣壳蛋白，L1和L2。 主要衣壳蛋白L1可以自发地组装成72-五聚体二十面体结构，其非常类似于天然病毒体。 虽然次要衣壳蛋白L2不是衣壳形成所必需的，但它被认为参与病毒基因组的衣壳化，并在病毒感染性进入途径中发挥许多重要作用。 L2的丰度及其在病毒粒子内的排列仍不清楚。冷冻电子显微镜和差异的3D重建分析纯化的衣壳揭示了二十面体有序的L2特定密度下的轴向管腔的每个L1壳粒。 此外，我们还研究了三种生化结构，试图确定HPV-16衣壳中含有多少L2。这些结果发表在J. Virology（2008）上。我们已经使用延时冷冻电子显微镜和图像分析来研究在293 T细胞中组装的HPV-16衣壳的成熟。主要的衣壳蛋白，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%;它的五聚体衣壳的结构发生了变化，最明显的是在它们的轴向区域;相邻衣壳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。不交联的C175 S突变体显示出类似的成熟相关的结构变化，但衣壳在其他类似条件下显著更大。我们的结论是，所观察到的结构尺寸的变化有利于成熟，但交联形成所需的锁定衣壳进入成熟状态。这些结果将提交出版。 我们还一直在开发计算工具，用于使用核磁共振数据研究生物大分子的结构和动力学。Xplor-NIH在以下领域继续开发：（a）进一步开发Python脚本接口，沿着大量文档;（B）能够根据纤维X射线衍射数据进行改进;（c）直接根据溶液X射线散射和小角度中子散射数据改进分子结构的额外工具和设施;（d）用于自动化核奥弗豪泽效应峰归属的PASD设施已得到实质性改进;（e）引入了新的结构细化目标，使得计算的蛋白质结构具有正确的分子密度。

The Imaging Sciences Laboratory has a major collaborative research effort with the 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. Ongoing research involves analyses of structures related to herpesvirus and papillomairus as well as other icosahedral virus capsids. Papillomaviruses (e.g. HPV-16) encode two capsid proteins, L1 and L2. The major capsid protein, L1, can assemble spontaneously into a 72-pentamer icosahedral structure that closely resembles native virions. Although the minor capsid protein L2 is not required for capsid formation, it is thought to participate in encapsidation of the viral genome, and plays a number of essential roles in the viral infectious entry pathway. The abundance of L2 and its arrangement within the virion remain unclear. Cryo-electron microscopy and difference 3D reconstruction analysis of purified capsids revealed an icosahedrally-ordered L2-specific density beneath the axial lumen of each L1 capsomer. In addition, we have studied three biochemical constructs to try to determine how much L2 is contained within HPV-16 capsids. These results were published in J. Virology (2008). We have used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV-16 capsids assembled in 293T cells. 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 capsomers change in structure, most markedly in their axial region; and the interaction surfaces between adjacent capsomers are consolidated. These structural changes are accompanied by the formation of disulfide crosslinks that enhance the stability of the mature capsid. The C175S mutant, which does not crosslink, shows similar maturation-related structural changes but capsids are significantly larger, under otherwise similar conditions. We conclude that the observed structural size changes facilitates maturation, but crosslink formation is required to lock the capsid into the mature state. These results will submitted for publication. We have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using NMR data. Development of Xplor-NIH has continued in the following areas: (a)further development of the Python scripting interface along with extensive documentation; (b) the ability to refine against fiber X-ray diffraction data; (c) additional tools and facilities for refining molecular structures directly against solution X-ray scattering and small-angle neutron scattering data; (d) the PASD facility for automated Nuclear Overhauser Effect peak assignment has been substantially improved; (e) a new structure refinement target has been introduced such that calculated protein structures have the correct molecular density.