Protein Structure Predictions, Atomic Model Building, and Validation Using a Cryo-EM Density Map from Hepatitis B Virus Spherical Subviral Particle.
Protein Structure Predictions, Atomic Model Building, and Validation Using a Cryo-EM Density Map from Hepatitis B Virus Spherical Subviral Particle.
复制标题
DOI：
10.21769/bioprotoc.4751
复制
发表时间：
2023-07-20
期刊：
影响因子：
0.8
通讯作者：
中科院分区：
文献类型：
--
作者：
乙型肝炎病毒（HBV）感染是全球公共卫生问题。B型肝炎病毒（HBV）感染是全球公共卫生问题。在慢性感染过程中，HBV小表面抗原以非感染性球形亚病毒颗粒（SVP）的形式大量过量表达，具有很强的免疫原性。迄今为止，对HBV球形SVP结构的理解仅限于12-30 μ m，关于其结构的结论相互矛盾。我们使用冷冻电子显微镜（cryo-EM）和3D图像重建将HBV球形SVP求解为6.3 μ m。在这里，我们提出了一个扩展协议，将AlphaFold 2预测与中等分辨率的cryo-EM密度图相结合，以建立可靠的3D模型。该方案使用了cryo-EM社区中常用的多个软件包。该工作流程包括3D模型预测、模型评估、刚体拟合、柔性拟合、真实空间细化、模型验证和模型调整。最后，所描述的协议也可以应用于高分辨率冷冻EM数据集（2-4 μ m）。
Hepatitis B virus (HBV) infection is a global public health concern. During chronic infection, the HBV small-surface antigen is expressed in large excess as non-infectious spherical subviral particles (SVPs), which possess strong immunogenicity. To date, attempts at understanding the structure of HBV spherical SVP have been restricted to 12–30 Å with contradictory conclusions regarding its architecture. We have used cryo-electron microscopy (cryo-EM) and 3D image reconstruction to solve the HBV spherical SVP to 6.3 Å. Here, we present an extended protocol on combining AlphaFold2 prediction with a moderate-resolution cryo-EM density map to build a reliable 3D model. This protocol utilizes multiple software packages that are routinely used in the cryo-EM community. The workflow includes 3D model prediction, model evaluation, rigid-body fitting, flexible fitting, real-space refinement, model validation, and model adjustment. Finally, the described protocol can also be applied to high-resolution cryo-EM datasets (2–4 Å).