Biomedical Imaging

成像科学实验室参与了与这些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。足以允许将L1晶体结构稳健地拟合到密度图中。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。这些结果将提交出版，并在新书的一章(Buck CB，trus BL(2012)《乳头瘤病毒病毒：一台隐藏分子跟腱的机器》中进行了部分讨论。在ADV Exp Med Biol 726：403-22中)。 我们一直在合作对含有病毒的RNA进行长期研究。在去年发表的一项研究(J Virol 86：6470-80)中，对兔出血症病毒(RHDV)的T=3衣壳结构进行了8A分辨率的研究。使用类似病毒的X射线结构，我们能够将(高分辨率)X射线结构拟合到EM密度中，从而创建一个伪原子模型。突变实验表明，N末端的变化可以改变VP1衣壳蛋白的包装、大小和对称性，从而产生T=4衣壳。在今年发表的另一项含有病毒的RNA研究中(J Virol 86：8314-8)，对双链RNA(DsRNA)病毒Cryphone ectria nitschkei Virus 1的冷冻电子显微镜重建显示，衣壳蛋白(60拷贝/颗粒)由重复的螺旋核心形成，表明基因复制。这种结构与L-A冠状病毒衣壳相似，提示有一个共同的120亚基T=1衣壳基序。 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。Xplor-NIH的开发继续在以下领域进行：(A)改进了处理SAXS和核磁共振RDC数据的方法，用于研究艾滋病毒衣壳蛋白的小寡聚体的混合物；(B)开发了用于研究淀粉样β结构的固体核磁共振方法；(C)大大改进了在蛋白质和核酸结构测定中普遍使用的统计势的使用。

The Imaging Sciences Laboratory is involved in 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 and complexed 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. 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 and are partially discussed in a new book chapter (Buck CB, Trus BL (2012) The papillomavirus virion: a machine built to hide molecular Achilles heels. In Adv Exp Med Biol 726:403-22). We have been collaborating in a long-term study of RNA containing viruses. In one study published last year (J Virol 86:6470-80) the T=3 capsid structure of Rabbit Hemorrhagic Disease Virus (RHDV) was studied to 8A resolution. Using X-ray structures from similar viruses, we were able to fit the (high-resolution) X-ray structure in the EM density creating a pseudo atomic model. Mutagenesis experiments were used to demonstrate that changes in the N terminus could change the packing, size, and symmetry of the VP1 capsid protein to produce T = 4 capsids. In another RNA containing virus study published this year (J Virol 86:8314-8) cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This structure is similar to totivirus L-A capsids suggesting a shared motif for 120-subunit T=1 capsids. We have also been developing computational tools for the study of the structure and dynamics of biological macromolecules using 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. Development of Xplor-NIH has continued in the following areas: (a) improved methods for treating SAXS and NMR RDC data in studying mixtures of small oligomers of theHIV capsid protein; (b) development of methods in solid state NMR applied to the study of amyloid beta structures; (c) much improved use of statistical potentials for general use in protein and nucleic acid structure determination.