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，以及分离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疱疹病毒、乳头瘤病毒和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。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。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。在略碱性的pH下，衣壳不能形成竞争性的二硫键(如成熟)。只需将裂解物缓冲到更中性的条件下，就可以生产具有90%二硫键的重组衣壳蛋白。冷冻-EM图像重建显示，与使用标准程序生产的HPV16衣壳相比，更成熟的重组HPV16衣壳显示出更大程度的规律性。它们更大的规律性使我们能够重建更高分辨率的衣壳(9)，足以使L1晶体结构稳健地适应密度图。生产完全成熟的重组衣壳的能力将有助于进一步研究天然HPV衣壳的结构。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。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。继续在以下领域开发Xplor-NIH结构确定软件包：(A)进一步开发Python脚本界面以及广泛的文件；(B)增加提炼能力 主要研究内容包括：(A)直接针对核磁共振松弛数据进行结构分析，具体用于确定蛋白质复合体的结构；(C)用于将SAXS和SANS数据纳入结构计算的新工具，适用于酶I蛋白质和酶I/HPR蛋白质复合体；(D)利用固体核磁共振实验数据的结构优化工具的应用，包括在结构确定中采用固体顺磁增强核磁共振数据和纤维衍射X射线数据。

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 in addition to structure determination and analysis of isolated proteins and nucleic acids. Ongoing research involves analyses of structures related to herpesvirus and papillomairus and 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. 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. 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. At slightly basic pH, the capsids do not achieve compete disulfide bond formation (e.g. maturation). Simply buffering the lysate to more neutral conditions allowed the production of recombinant capsids with >90% disulfide bonds. Cryo-EM with image reconstruction revealed that more fully mature recombinant HPV16 capsids exhibited a much greater degree of regularity compared to HPV16 capsids produced using standard procedures. Their greater regularity allowed us to reconstruct the capsid to higher resolution ( 9), sufficient to allow robust fitting of the L1 crystal structure into the density map. 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. 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 the Xplor-NIH software package for structure determination has continued in the following areas: (a) further development of the Python scripting interface along with extensive documentation; (b) the addition of the ability to refine directly against NMR relaxation data, with the specific application to protein complex structure determination; (c) new tools for incorporating SAXS and SANS data into structure calculations, applied to the Enzyme I protein and Enzyme I/HPr protein complex; (d) application of structure refinement tools using data from Solid-State NMR experiments, including the incorporation of solid state paramagnetic enhancement NMR data and fiber diffraction X-ray data in structure determination.