Lugmayr Wolfgang, Kotov Vadim, Goessweiner-Mohr Nikolaus, Wald Jiri, DiMaio Frank, Marlovits Thomas C
University Medical Center Hamburg-Eppendorf (UKE), Institute of Structural and Systems Biology, Hamburg, Germany.
CSSB Centre for Structural Systems Biology, Hamburg, Germany.
Nat Protoc. 2023 Jan;18(1):239-264. doi: 10.1038/s41596-022-00757-9. Epub 2022 Nov 2.
Cryogenic electron microscopy (cryo-EM) data represent density maps of macromolecular systems at atomic or near-atomic resolution. However, building and refining 3D atomic models by using data from cryo-EM maps is not straightforward and requires significant hands-on experience and manual intervention. We recently developed StarMap, an easy-to-use interface between the popular structural display program ChimeraX and Rosetta, a powerful molecular modeling engine. StarMap offers a general approach for refining structural models of biological macromolecules into cryo-EM density maps by combining Monte Carlo sampling with local density-guided optimization, Rosetta-based all-atom refinement and real-space B-factor calculations in a straightforward workflow. StarMap includes options for structural symmetry, local refinements and independent model validation. The overall quality of the refinement and the structure resolution is then assessed via analytical outputs, such as magnification calibration (pixel size calibration) and Fourier shell correlations. Z-scores reported by StarMap provide an easily interpretable indicator of the goodness of fit for each residue and can be plotted to evaluate structural models and improve local residue refinements, as well as to identify flexible regions and potentially functional sites in large macromolecular complexes. The protocol requires general computer skills, without the need for coding expertise, because most parts of the workflow can be operated by clicking tabs within the ChimeraX graphical user interface. Time requirements for the model refinement depend on the size and quality of the input data; however, this step can typically be completed within 1 d. The analytical parts of the workflow are completed within minutes.
低温电子显微镜（cryo-EM）数据呈现出大分子系统在原子或近原子分辨率下的密度图。然而，利用cryo-EM图数据构建和完善三维原子模型并非易事，需要大量的实际操作经验和人工干预。我们最近开发了StarMap，它是广受欢迎的结构显示程序ChimeraX与强大的分子建模引擎Rosetta之间一个易于使用的接口。StarMap通过在一个简单的工作流程中结合蒙特卡罗采样与局部密度引导优化、基于Rosetta的全原子精修以及实空间B因子计算，提供了一种将生物大分子结构模型精修到cryo-EM密度图中的通用方法。StarMap包括结构对称性、局部精修和独立模型验证的选项。然后通过分析输出，如放大率校准（像素大小校准）和傅里叶壳层相关，来评估精修的整体质量和结构分辨率。StarMap报告的Z分数为每个残基的拟合优度提供了一个易于解释的指标，可以绘制出来以评估结构模型并改进局部残基精修，以及识别大型大分子复合物中的柔性区域和潜在功能位点。该方案只需要一般的计算机技能，无需编码专业知识，因为工作流程的大部分操作都可以通过在ChimeraX图形用户界面中点击标签来完成。模型精修的时间要求取决于输入数据的大小和质量；然而，这一步通常可以在1天内完成。工作流程的分析部分在几分钟内即可完成。
