Koteiche Hanane A, Chiu Steve, Majdoch Rebecca L, Stewart Phoebe L, Mchaourab Hassane S
Department of Molecular Physiology and Biophysics, Center for Structural Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Structure. 2005 Aug;13(8):1165-71. doi: 10.1016/j.str.2005.05.006.
We report an approach for determining the structure of macromolecular assemblies by the combined application of cryo-electron microscopy (cryo-EM) and site-directed spin labeling electron paramagnetic resonance spectroscopy (EPR). This approach is illustrated for Hsp16.5, a small heat shock protein that prevents the aggregation of nonnative proteins. The structure of Hsp16.5 has been previously studied by both cryo-EM and X-ray crystallography. The crystal structure revealed a roughly spherical protein shell with dodecameric symmetry; however, residues 1-32 were found to be disordered. The cryo-EM reconstruction at 13 A resolution appeared similar to the crystal structure but with additional internal density corresponding to the N-terminal regions of the 24 subunits. In this study, a systematic application of site-directed spin labeling and EPR spectroscopy was carried out. By combining the EPR constraints from spin label accessibilities and proximities with the cryo-EM density, we obtained an atomic model for a portion of the Hsp16.5 N-terminal region in the context of the oligomeric complex.
我们报道了一种通过联合应用冷冻电子显微镜（cryo-EM）和定点自旋标记电子顺磁共振波谱（EPR）来确定大分子组装体结构的方法。以Hsp16.5为例说明了这种方法，Hsp16.5是一种防止非天然蛋白质聚集的小热休克蛋白。Hsp16.5的结构先前已通过冷冻电子显微镜和X射线晶体学进行了研究。晶体结构揭示了一个具有十二聚体对称性的大致球形蛋白质外壳；然而，发现第1至32位残基是无序的。13埃分辨率的冷冻电子显微镜重建结果看起来与晶体结构相似，但有对应于24个亚基N端区域的额外内部密度。在本研究中，进行了定点自旋标记和EPR波谱的系统应用。通过将来自自旋标记可及性和邻近性的EPR限制与冷冻电子显微镜密度相结合，我们在寡聚复合物的背景下获得了Hsp16.5 N端区域一部分的原子模型。
