RECOGNITION BY RECEPTORS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 病原体和宿主之间相互作用的特异性允许入侵（当病原体识别宿主时）和免疫识别（当宿主识别病原体时）。这些项目研究两种类型的相互作用，包括跨膜区域以及分离的蛋白质可溶性区域。最初的结构尚未公布。 第一个项目包括人类Toll样受体（TLR）和细菌外膜孔蛋白之间的相互作用。我们已经生长出了细菌孔蛋白的初始晶体，这些细菌孔蛋白是TLR配体，其分辨率可达2.3？。这些晶体在三个维度中的两个维度上都很小，使得大多数具有可调谐波长（>0.1 mm）的同步加速器的大光束尺寸限制了我们数据的质量。我们无法看到任何衍射使用我们的家庭发电机。由于这些孔蛋白与已知结构的孔蛋白不具有显著的序列相似性，因此分子置换失败。该蛋白没有足够的蛋氨酸来执行Se-Met MAD，我们正在收集衍生物。从迄今为止收集的50个数据集中，我们已经确定了两个衍生物。第一个是一个Lu衍生物与一个单一的网站，并在此beamtime，我们建议做一个完整的4波长MAD的Lu浸泡晶体，以改善的阶段。第二衍生物是具有4个位点的碘化物衍生物。这些晶体衍射较差（4 A分辨率），我们建议引入改变浸泡浓度和时间的晶体，以将衍射极限提高到优于3.5 A分辨率。此外，我们将带来其他几个重原子浸泡晶体（见上文）。 第二个项目研究相反的病原体识别现象--即细菌受体对宿主唾液酸的识别。已经生长了细菌受体（GspB）的识别区的晶体。像孔蛋白晶体一样，它们生长为细棒，缺乏足够的蛋氨酸来确定Se-Met的结构。此外，我们认为这些晶体的小尺寸排除了在我们的家用发电机上观察到任何衍射。我们正处于寻找重原子衍生物的开始阶段;虽然已经从浸泡了5种不同化合物的晶体中收集了数据，但尚未发现任何衍生物。我们将带来天然蛋白质，以确定更小的光束尺寸是否会提高数据集的质量，此外，我们将带来上述衍生浸泡用于MAD数据收集。 最后一个项目是完整的膜细菌复合物II同系物醌醇：延胡索酸还原酶。这些晶体生长为长棒。在家用发生器上收集的最佳数据集是6A分辨率，而在同步加速器上收集的最佳数据集是2.7A分辨率。我们目前的项目是确定与伴侣蛋白的共结构。迄今为止，文献中仅报道了一种膜蛋白与伴侣蛋白复合的共结构。因此，从技术发展的角度来看，这种结构的意义将是巨大的。此外，这种复合体的生物学意义是巨大的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The specificity of interactions between pathogen and host allows both invasion (when the pathogen recognizes the host) and immunological recognition (when the host recognizes the pathogen. These projects investigate both types of interactions, and include both the membrane-spanning region as well as the separated soluble region of the proteins. The initial structures have not yet been published. The first project encompasses the interaction between human toll like receptors (TLRs) and bacterial outer membrane porins. We have grown initial crystals of the bacterial porins that are TLR ligands that diffract to 2.3 ¿ resolution. These crystals are small in two of the three dimensions such that the large beam size at most synchrotrons with tunable wavelengths (>0.1 mm) limits the quality of our data. We are unable to see any diffraction using our home generator. Since these porins do not have significant sequence similarity to porins of known structure, molecular replacement has failed. The protein does not have sufficient methionines to perform Se-Met MAD and we are collecting derivatives. From 50 data sets collected so far, we have identified two derivatives. The first is a Lu derivative with a single site, and during this beamtime, we propose to do a full 4-wavelength MAD on Lu-soaked crystals in order to improve the phases. The second derivative is an iodide derivative with 4 sites. These crystals diffracted poorly (4 A resolution) and we propose bringing crystals that vary the soaking concentration and times in order to improve the diffraction limit to better than 3.5 A resolution. In addition, we will bring several other heavy atom soaked crystals (see above). The second project investigates the opposite pathogenic recognition phenomenon - i.e. the recognition of host siaylic acids by the bacterial receptor. Crystals have been grown of the recognition region of the bacterial receptor (GspB). Like the porin crystals, these grow as thin rods and lack sufficient methionines to determine the structure by Se-Met. In addition, we believe that the small size of these crystals is precluding observation of any diffraction on our home generator. We are at the beginning of searching for heavy atom derivatives; although data have been collected from crystals soaked with 5 distinct compounds, no derivatives have yet been identified. We will bring native protein to determine if a smaller beam size will improve the quality of the data set, and in addition, we will bring the above mentioned derivative soaks for MAD data collection. The final project is of the integral-membrane bacterial complex II homolog quinol:fumarate reductase. These crystals grow as long rods. The best data set collected on the home generator is at 6A resolution, while the best data set collected at the synchrotron is at 2.7 A resolution. Our current project is to determine a co-structure with a partner protein. Only one co-structure of membrane proteins in complex with a partner protein has been reported in the literature to date. Thus the significance of this structure from a technique development standpoint would be great. In addition, the biological significance of this complex is immense.