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StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement

Nature Protocols volume 18, pages 239–264 (2023)Cite this article

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Abstract

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.

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Fig. 1: StarMap/Rosetta pipeline.
Fig. 2: Screenshot of the StarMap GUI.
Fig. 3: Molecular symmetry supported by StarMap.
Fig. 4: Exemplary run of the StarMap/Rosetta pipeline.
Fig. 5: Analysis of a deposited model in StarMap.
Fig. 6: Handling ligands in StarMap.
Fig. 7: Job execution options in StarMap.

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Data availability

The example workflow (Box 3) uses a deposited map (EMDB entry 13054), and the corresponding protein sequence is obtained from UniProt86 (accession number B7UMA7). All required input and output files are provided in Supplementary Data 1.

Code availability

StarMap is available under libre/open source license (BSD 2-Clause ‘Simplified’ License) from GitHub (https://github.com/wlugmayr/chimerax-starmap). StarMap version 1.1.66 used to generate data from Box 3 is available from the Supplement.

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Acknowledgements

We thank all current and former members of the Marlovits laboratory for their support in this project. High-performance computing was possible through access to the computing clusters at Deutsches Elektronen Synchrotron (DESY)/Hamburg (Germany) and the Vienna Scientific Cluster (Austria). This project was supported by funds available to T.C.M. through the Behörde für Wissenschaft, Forschung und Gleichstellung of the city of Hamburg at the Institute of Structural and Systems Biology at the University Medical Center Hamburg–Eppendorf (UKE), DESY, the Institute for Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences and the Research Institute of Molecular Pathology (IMP).

Author information

Author notes

  1. Vadim Kotov

    Present address: Evotec SE, Hamburg, Germany

  2. Nikolaus Goessweiner-Mohr

    Present address: Johannes Kepler University, Institute of Biophysics, Linz, Austria

  3. These authors contributed equally: Wolfgang Lugmayr, Vadim Kotov.

Authors and Affiliations

  1. University Medical Center Hamburg-Eppendorf (UKE), Institute of Structural and Systems Biology, Hamburg, Germany

    Wolfgang Lugmayr, Vadim Kotov, Nikolaus Goessweiner-Mohr, Jiri Wald & Thomas C. Marlovits

  2. CSSB Centre for Structural Systems Biology, Hamburg, Germany

    Wolfgang Lugmayr, Vadim Kotov, Nikolaus Goessweiner-Mohr, Jiri Wald & Thomas C. Marlovits

  3. Deutsches Elektronen Synchrotron (DESY), Hamburg, Germany

    Wolfgang Lugmayr, Vadim Kotov, Nikolaus Goessweiner-Mohr, Jiri Wald & Thomas C. Marlovits

  4. Research Institute of Molecular Pathology (IMP), Vienna, Austria

    Wolfgang Lugmayr, Vadim Kotov, Nikolaus Goessweiner-Mohr, Jiri Wald & Thomas C. Marlovits

  5. Institute for Molecular Biotechnology (IMBA), Austrian Academy of Sciences, Vienna, Austria

    Wolfgang Lugmayr, Vadim Kotov, Nikolaus Goessweiner-Mohr, Jiri Wald & Thomas C. Marlovits

  6. University of Washington, Department of Biochemistry, Seattle, WA, USA

    Frank DiMaio

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Contributions

Conceptualization: F.D.M. and T.C.M. Methodology: W.L., F.D.M. and T.C.M. Software: W.L. and F.D.M. Investigation: N.G.M., J.W., V.K. Formal analysis: all authors. Resources: T.C.M. Writing—original draft: V.K. and W.L. Writing—review and editing: all authors. Visualization: V.K. and W.L. Supervision: T.C.M. Funding acquisition: T.C.M.

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Correspondence to Thomas C. Marlovits.

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Key papers related to this protocol

DiMaio, F. et al. Nat. Methods 12, 361–365 (2015): https://doi.org/10.1038/nmeth.3286

Wang, R. Y.-R. et al. eLife 5, e17219 (2016): https://doi.org/10.7554/eLife.17219

Kotov, V. et al. Biochem. Biophys. Rep. 27, 101039 (2021): https://doi.org/10.1016/j.bbrep.2021.101039

Wald, J. et al. Nature 609, 630–639 (2022): https://doi.org/10.1038/s41586-022-05121-1

Supplementary information

Supplementary Video 1

This short video shows how to set up a StarMap refinement and how to inspect outputs

Supplementary Software 1

A distribution of StarMap (any platform)

Supplementary Data 1

Input and output files for an exemplary StarMap run (Box 3)

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Lugmayr, W., Kotov, V., Goessweiner-Mohr, N. et al. StarMap: a user-friendly workflow for Rosetta-driven molecular structure refinement. Nat Protoc 18, 239–264 (2023). https://doi.org/10.1038/s41596-022-00757-9

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