Biomedical Imaging

成像科学实验室与这些研究所开展了一项重要的合作研究工作，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照片和核磁共振谱，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。正在进行的研究包括分析与疱疹病毒和乳头瘤病毒以及其他二十面体病毒衣壳有关的结构。 乳头瘤病毒(如HPV-16)编码两种衣壳蛋白L1和L2。主要衣壳蛋白L1可以自发组装成一个72个五聚体的二十面体结构，与天然病毒粒子非常相似。虽然次要衣壳蛋白L2不是衣壳形成所必需的，但它被认为参与了病毒基因组的囊化，并在病毒感染进入途径中发挥了许多重要作用。L2的丰度及其在病毒粒子中的排列仍不清楚。冷冻电子显微镜和差值三维重建分析显示，每个L1胶囊的轴向内腔下都有一个二十面体有序的L2特定密度。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《病毒学杂志》(2008)上。我们用时移低温电子显微镜和图像分析技术研究了293T细胞中组装的HPV-16衣壳的成熟过程。主要的衣壳蛋白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，proapsid的直径缩小了5%；它的五聚体胶囊结构发生了变化，最明显的是在其轴向区域；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。C175S突变体没有交联，表现出类似的成熟相关结构变化，但在其他类似的条件下，衣壳明显更大。我们的结论是，观察到的结构尺寸变化有助于成熟，但需要交联剂的形成才能将衣壳锁定到成熟状态。这些结果将提交发表。 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。Xplor-NIH的开发在下列领域继续进行：(A)进一步开发Python脚本界面以及广泛的文件；(B)能够针对纤维X射线衍射数据进行优化；(C)更多工具和设施用于直接针对溶液X射线散射和小角中子散射数据优化分子结构；(D)用于自动核Overhauser效应峰分配的PASD设施已大幅改进；(E)引入了一个新的结构优化靶标，可以计算出具有正确分子密度的蛋白质结构。

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.