Three-dimensional Structures Of Biological Macromolecules

生物大分子 传统的分子建模是在原子分辨率下进行的，这依赖于X射线和NMR实验来提供结构信息。 当处理数百万原子的生物分子组装体时，分子对象的原子描述变得非常计算低效。 我们开发了一种方法，使用地图对象的分子建模，有效地从实验地图中获得结构信息，以及方便地操纵地图对象，直接使用地图对象进行构象搜索。 这一开发工作已经作为EMAP模块在CHARMM中实现。 该实现使CHARMM能够操纵地图对象，包括地图输入、输出、比较、对接等。其他实验如过渡金属离子FRET（tmFRET）正在成为获得蛋白质结构信息的有用方法。 将联合收割机高效的模拟技术与实验获得的结构信息相结合，辅助高通量蛋白质结构测定是我们研究的一个新热点。 从低分辨率电磁图确定构造 我们开发了一个地图约束的自我引导Langevin动力学（MapSGLD）模拟方法，有效的有针对性的构象搜索。有针对性的构象搜索表示在由实验观察和/或由用户指定的结构要求限定的约束下的模拟。通过地图约束，该方法提供了一种有效的方法来维护子结构和结构目标的构象搜索。由于自导引Langevin动力学具有更强的构象搜索能力，该方法适用于模拟大规模的构象变化，如大分子组装体的形成和不同构象状态之间的转变.这种方法的一个直接应用是通过将原子结构灵活地拟合到来自低温电子显微镜的密度图中来确定大分子结构。 通过X射线与cyro-EM识别的沃茨位置的分析。 通过冷冻EM和X射线晶体学确定的半乳糖苷酶原子模型中确定的水分子之间的相关性。这项工作是与美国国立卫生研究院国家癌症研究所的冷冻EM实验室合作进行的。它是基于他们最近发表的2.2-分辨率解决方案结构的-半乳糖苷酶，其中包含解决水密度。这项研究分析了如何通过冷冻EM确定的水的位置比较保守的水在所有晶体学确定的结构。水分子和氨基酸之间的相互作用发生在蛋白质表面和结构内，包括亚基界面，催化位点和其他空腔等区域。识别蛋白质溶剂化特征对于理解蛋白质结构和功能以及设计用于药物发现的高亲和力先导化合物至关重要。 蛋白质结合位点的全球组织。 通过构建基于结构相似性的所有已知结合位点的加权网络，并基于最小描述长度原则检测结构相似结合位点的社区，来获得蛋白质结合位点的全局组织。分析表明，有两个中心结合位点社区，它们扮演着较小外围社区的网络枢纽的角色。社区的大小遵循幂律分布，这表明较大社区中包含的结合位点可能更古老，并且是最近的进化结构支架。在相同的社区结构相似的结合位点结合到不同的配体混杂，他们也嵌入在不同的结构域结构。了解结合位点相互作用的一般原理将为改进药物设计和蛋白质设计铺平道路。 谷氨酸受体激活的结构机制 离子型谷氨酸受体是阳离子通道，其通过响应于从突触前神经元释放的L-谷氨酸而使突触后膜去极化来介导信号传递。 在iGluR受体家族中有α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPA）、钾盐镁矾（KA）和N-甲基-D-天冬氨酸（NMDA）亚型，这些受体都被谷氨酸激活并且在氨基酸序列上相关，但在总体结构、药理学和生物物理特征上不同。 AMPA受体是由具有模块化结构域排列的亚基组成的四聚体复合物，从氨基末端结构域（ATD）、配体或激动剂结合结构域（LBD）和成孔跨膜结构域（TMD）开始。 由于AMPA受体在激动剂的持续存在下经历快速且几乎完全的脱敏，因此已经证明难以阐明激动剂结合的活化状态的高分辨率结构，并且难以定义激动剂结合的化学势被转换成离子通道门控的机械力的机制。 我们以前开发的地图约束自引导Langevin动力学（MapSGLD）模拟方法可以利用嵌入在力场中的结构信息，灵活地将大分子系统拟合到低分辨率地图中，以获得满足地图的能量有利的原子结构。我们使用MapSGLD进行灵活的拟合，从EM图中获得谷氨酸受体的原子结构。 谷氨酸受体的开放态原子结构显示出与LBD X射线结构一致的蛤壳闭合构象中的LBD。 除了结构确定之外，MapSGLD还提供有关不同状态之间转换的动态信息。

Biological Macromolecules Traditional molecular modeling is performed at atomic resolution, which relies on X-ray and NMR experiments to provide structural information. When dealing with biomolecular assemblies of millions of atoms, atomic description of molecular objects becomes very computational inefficient. We developed a method that uses map objects for molecular modeling to efficiently derive structural information from experimental maps, as well as conveniently manipulate map objects, perform conformational search directly using map objects. This development work has been implemented into CHARMM as the EMAP module. This implementation enables CHARMM to manipulate map objects, including map input, output, comparison, docking, etc. Other experiment such as transition metal ion FRET (tmFRET) is becoming a useful way to obtain protein structure information. A new focus of our research is to combine efficient simulation technique with structural information from experiment to assist high throughput protein structure determination. Structure determination from low resolution EM maps We developed a map-restrained self-guided Langevin dynamics (MapSGLD) simulation method for efficient targeted conformational search. The targeted conformational search represents simulations under restraints defined by experimental observations and/or by user specified structural requirements. Through map-restraints, this method provides an efficient way to maintain substructures and to set structure targets during conformational searching. With an enhanced conformational searching ability of self-guided Langevin dynamics, this approach is suitable for simulating large-scale conformational changes, such as the formation of macromolecular assemblies and transitions between different conformational states. A direct application of this method is to determine macromolecular structures by flexible fitting of atomic structures into density maps derived from cryo-electron microscopy. Analysis of waters positions identified by X-ray versus cyro-EM. Correlation between water molecules identified in atomic models of -galactosidase determined by cryo-EM and X-ray crystallography. This work is a collaboration with a cryo-EM laboratory at the National Cancer institute, NIH. It is based on their recently published 2.2- resolution solution structure of -galactosidase, that contains resolved water densities. This study analyses how the water positions determined by cryo-EM compare to conserved water across all crystallography-determined structures. Interactions between water molecules and amino acids occur both at the surface of protein and within the structure, including areas such as subunit interfaces, catalytic sites, and other cavities. Identifying protein solvation profiles are critical to understand both protein structure and function, as well as for the design of high affinity lead compounds for drug discovery. The global organization of protein binding sites. A global organization of protein binding sites is obtained by constructing a weighted network of all known binding sites based on their structural similarities and detecting communities of structurally similar binding sites based on the minimum description length principle. The analysis reveals that there are two central binding site communities that play the roles of the network hubs of smaller peripheral communities. The sizes of communities follow a power-law distribution, which indicates that the binding sites included in larger communities may be older and have been evolutionary structural scaffolds of more recent ones. Structurally similar binding sites in the same community bind to diverse ligands promiscuously and they are also embedded in diverse domain structures. Understanding the general principles of binding site interplay will pave the way for improved drug design and protein design. Structure mechanism of Glutamate receptor activation Ionotropic glutamate receptors are cation channels that mediate signal transmission by depolarizing the postsynapitic membrane in response to L-glutamate release from the presynaptic neuron. Within the iGluR family of receptors are a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), kainite(KA), and N-methyl-D-aspartate (NMDA) subtypes, receptors that are all activated by glutamate and related in amino acid sequence, yet distinct in overall architecture, pharmacology, and biophysical characteristics. AMPA receptors are tetrameric complexes composed of subunits with a modular domain arrangement, beginning with the amino-terminal domain(ATD), the ligand- or agonist-binding domain (LBD), and the pore-forming transmembrane domain (TMD). Because AMPA receptors undergo rapid and nearly complete desensitization in the continued presence of agonist, it has proven difficult to elucidate high-resolution structures of agonist-bound, activated states and to define mechanism by which the chemical potential of agonist binding is transduced into the mechanical force of ion channel gating. The map-restrained self-guided Langevin dynamics (MapSGLD) simulation method we developed previously can utilize structural information embedded in a force field to flexibly fit macromolecular systems into low resolution maps to obtain energetically favored atomic structures that satisfy the maps. We perform flexible fitting with MapSGLD to obtain atomic structures of the glutamate receptor from EM maps. The open state atomic structure of the glutamate receptor shows the LBD in the clamshell closed conformation that agrees with the LBD x-ray structure. In addition to structural determination, MapSGLD provides dynamic information about the transition between different states.