Skip to main content

Advertisement

Log in

Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

Abstract

Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1: Flowchart illustrating the main steps to obtain optimally thermostabilized GPCR mutants tailored for successful crystallization in LCP.
Fig. 2: Effects of different antagonists on SEC elution profiles, the thermal stability in the CPM assay, and the mobile fraction in FRAP experiments, exemplified by theophylline- and ZM241385-bound A2AR (A2AR-bRIL-∆C, Protein Data Bank ID: 4EIY).
Fig. 3: CPM-based thermostabilization of NK1R-y04 in an antagonist-bound conformation.
Fig. 4: Modular SLIC-based cloning and construct generation toolbox.
Fig. 5: CPM-based screening of fusion proteins and insertion positions to replace the ICL3 of NK1R-S for crystallization in LCP.
Fig. 6: Screening and engineering of a successful crystallization construct of the human OTR.
Fig. 7: CPM-based thermostabilization of PTH1R in an agonist-bound conformation and screening of fusion proteins and their insertion positions.

Similar content being viewed by others

Data availability

All data needed to evaluate the conclusions on the paper are present in the paper. The datasets generated and analyzed here are available from the authors upon request.

References

  1. Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov. 16, 829–842 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Katritch, V., Cherezov, V. & Stevens, R. C. Structure–function of the G protein–coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Tesmer, J. J. G. Hitchhiking on the heptahelical highway: structure and function of 7TM receptor complexes. Nat. Rev. Mol. Cell Biol. 17, 439–450 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Xiang, J. et al. Successful strategies to determine high-resolution structures of GPCRs. Trends Pharmacol. Sci. 37, 1055–1069 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Erlandson, S. C., McMahon, C. & Kruse, A. C. Structural basis for G protein–coupled receptor signaling. Annu. Rev. Biophys. 47, 1–18 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Cao, C., Zhang, H., Yang, Z. & Wu, B. Peptide recognition, signaling and modulation of class B G protein-coupled receptors. Curr. Opin. Chem. Biol. 51, 53–60 (2018).

    CAS  Google Scholar 

  7. Thal, D. M., Glukhova, A., Sexton, P. M. & Christopoulos, A. Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018).

    Article  CAS  PubMed  Google Scholar 

  8. Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Aci. USA 105, 877–882 (2008).

    Article  CAS  Google Scholar 

  9. Maeda, S. & Schertler, G. F. X. Production of GPCR and GPCR complexes for structure determination. Curr. Opin. Struc. Biol. 23, 381–392 (2013).

    Article  CAS  Google Scholar 

  10. Rosenbaum, D. M., Rasmussen, S. G. F. & Kobilka, B. K. The structure and function of G-protein-coupled receptors. Nature 459, 356–363 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kobilka, B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271 (1995).

    Article  CAS  PubMed  Google Scholar 

  12. Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sarkar, C. A. et al. Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc.Natl Acad. Aci. USA 105, 14808–14813 (2008).

    Article  CAS  Google Scholar 

  15. Schlinkmann, K. M. et al. Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution. J. Mol. Biol. 422, 414–428 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Scott, D. J. & Plückthun, A. Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells. J. Mol. Biol. 425, 662–677 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Schütz, M. et al. Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts. Sci. Rep. 6, 21508 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Magnani, F., Shibata, Y., Serrano-Vega, M. J. & Tate, C. G. Co-evolving stability and conformational homogeneity of the human adenosine A2A receptor. Proc. Natl Acad. Sci. USA 105, 10744–10749 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tate, C. G. A crystal clear solution for determining G-protein-coupled receptor structures. Trends Biochem. Sci. 37, 343–352 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Zhang, X., Stevens, R. C. & Xu, F. The importance of ligands for G protein-coupled receptor stability. Trends Biochem. Sci. 40, 79–87 (2015).

    Article  PubMed  Google Scholar 

  21. Miller, R. L. et al. The importance of ligand-receptor conformational pairs in stabilization: spotlight on the N/OFQ G protein-coupled receptor. Structure 23, 2291–2299 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Glukhova, A. et al. Structure of the adenosine A1 receptor reveals the basis for subtype selectivity. Cell 168, 867–877.e13 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762.e14 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ma, Y. et al. Structural basis for apelin control of the human apelin receptor. Structure 25, 858–866.e4 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Cherezov, V. & Caffrey, M. Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins. J. Appl. Crystallogr. 36, 1372–1377 (2003).

    Article  CAS  Google Scholar 

  26. Cherezov, V. Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin. Struc. Biol. 21, 559–566 (2011).

    Article  CAS  Google Scholar 

  27. Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F. & Caffrey, M. A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr. D. Biol. Crystallogr. 60, 1795–1807 (2004).

    Article  PubMed  Google Scholar 

  28. Magnani, F. et al. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies. Nat. Protoc. 11, 1554–1571 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Waltenspühl, Y., Ehrenmann, J., Klenk, C. & Plückthun, A. Engineering of challenging G protein-coupled receptors for structure determination and biophysical studies. Molecules 26, 1465 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Schöppe, J. et al. Crystal structures of the human neurokinin 1 receptor in complex with clinically used antagonists. Nat. Commun. 10, 17 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Waltenspühl, Y., Schöppe, J., Ehrenmann, J., Kummer, L. & Plückthun, A. Crystal structure of the human oxytocin receptor. Sci. Adv. 6, eabb5419 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ehrenmann, J. et al. High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist. Nat. Struct. Mol. Biol. 25, 1086–1092 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Alexandrov, A. I., Mileni, M., Chien, E. Y., Hanson, M. A. & Stevens, R. C. Microscale fluorescent thermal stability assay for membrane proteins. Structure 16, 351–359 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Ayers, F. C., Warner, G. L., Smith, K. L. & Lawrence, D. A. Fluorometric quantitation of cellular and nonprotein thiols. Anal. Biochem. 154, 186–193 (1986).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, Z., Ye, C., Zhang, X. & Wei, Y. Cysteine residue is not essential for CPM protein thermal-stability assay. Anal. Bioanal. Chem. 407, 3683–3691 (2015).

    Article  CAS  PubMed  Google Scholar 

  36. Cherezov, V., Liu, J., Griffith, M., Hanson, M. A. & Stevens, R. C. LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization. Cryst. Growth Des. 8, 4307–4315 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chien, E. Y. T. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330, 1091–1095 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Thompson, A. A. et al. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485, 395–399 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Manglik, A. et al. Crystal structure of the µ-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wu, H. et al. Structure of the human κ-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang, C. et al. Structure of the human smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wacker, D. et al. Structural features for functional selectivity at serotonin receptors. Science 340, 615–619 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang, C. et al. Structural basis for molecular recognition at serotonin receptors. Science 340, 610–614 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Tan, Q. et al. Structure of the CCR5 chemokine receptor–HIV entry inhibitor maraviroc complex. Science 341, 1387–1390 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Wu, H. et al. Structure of a class C GPCR metabotropic glutamate receptor 1 bound to an allosteric modulator. Science 344, 58–64 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang, J. et al. Agonist-bound structure of the human P2Y12 receptor. Nature 509, 119–122 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chrencik, J. E. et al. Crystal structure of antagonist bound human lysophosphatidic acid receptor 1. Cell 161, 1633–1643 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zheng, Y. et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540, 458–461 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Song, G. et al. Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators. Nature 546, 312–315 (2017).

    Article  CAS  PubMed  Google Scholar 

  52. Wang, S. et al. Structure of the D2 dopamine receptor bound to the atypical antipsychotic drug risperidone. Nature 555, 269–273 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Peng, Y. et al. 5-HT2C receptor structures reveal the structural basis of GPCR polypharmacology. Cell 172, 719–730.e14 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Cao, C. et al. Structural basis for signal recognition and transduction by platelet-activating-factor receptor. Nat. Struct. Mol. Biol. 25, 488–495 (2018).

    Article  CAS  PubMed  Google Scholar 

  55. Claff, T. et al. Elucidating the active δ-opioid receptor crystal structure with peptide and small-molecule agonists. Sci. Adv. 5, eaax9115 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Gusach, A. et al. Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors. Nat. Commun. 10, 5573 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Johansson, L. C. et al. XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity. Nature 569, 289–292 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Luginina, A. et al. Structure-based mechanism of cysteinyl leukotriene receptor inhibition by antiasthmatic drugs. Sci. Adv. 5, eaax2518 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Stauch, B. et al. Structural basis of ligand recognition at the human MT1 melatonin receptor. Nature 569, 284–288 (2019).

    Article  CAS  PubMed  Google Scholar 

  60. Toyoda, Y. et al. Ligand binding to human prostaglandin E receptor EP4 at the lipid–bilayer interface. Nat. Chem. Biol. 15, 18–26 (2019).

    Article  CAS  PubMed  Google Scholar 

  61. White, K. L. et al. Structural connection between activation microswitch and allosteric sodium site in GPCR signaling. Structure 26, 259–269.e5 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Yu, J. et al. Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding. Science 368, 428–433 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Xu, F., Liu, W., Hanson, M. A., Stevens, R. C. & Cherezov, V. Development of an automated high throughput LCP-FRAP assay to guide membrane protein crystallization in lipid mesophases. Cryst. Growth Des. 11, 1193–1201 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Li, M. Z. & Elledge, S. J. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat. Methods 4, 251–256 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Kuzminov, A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage λ. Microbiol. Mol. Biol. Rev. 63, 751–813 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Smith, H. O. & Wilcox, K. W. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. Mol. Biol. 51, 379–391 (1970).

    Article  CAS  PubMed  Google Scholar 

  67. Gay, P., Le Coq, D., Steinmetz, M., Berkelman, T. & Kado, C. I. Positive selection procedure for entrapment of insertion sequence elements in Gram-negative bacteria. J. Bacteriol. 164, 918–921 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Ghosh, E., Kumari, P., Jaiman, D. & Shukla, A. K. Methodological advances: the unsung heroes of the GPCR structural revolution. Nat. Rev. Mol. Cell Biol. 16, 69–81 (2015).

    Article  CAS  PubMed  Google Scholar 

  69. Safarik, I. & Safarikova, M. Magnetic techniques for the isolation and purification of proteins and peptides. BioMagn. Res. Technol. 2, 7 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  70. Brizzard, B. L., Chubet, R. G. & Vizard, D. L. Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. BioTechniques 16, 730–735 (1994).

    CAS  PubMed  Google Scholar 

  71. Morrison, K. L. & Weiss, G. A. Combinatorial alanine-scanning. Curr. Opin. Chem. Biol. 5, 302–307 (2001).

    Article  CAS  PubMed  Google Scholar 

  72. Faham, S. et al. Side-chain contributions to membrane protein structure and stability. J. Mol. Biol. 335, 297–305 (2004).

    Article  CAS  PubMed  Google Scholar 

  73. Yin, J., Mobarec, J. C., Kolb, P. & Rosenbaum, D. M. Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant. Nature 519, 247–250 (2015).

    Article  CAS  PubMed  Google Scholar 

  74. Lebon, G., Bennett, K., Jazayeri, A. & Tate, C. G. Thermostabilisation of an agonist-bound conformation of the human adenosine A2A receptor. J. Mol. Biol. 409, 298–310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Mirzadegan, T., Benkö, G., Filipek, S. & Palczewski, K. Sequence analyses of G-protein-coupled receptors: similarities to rhodopsin. Biochemistry 42, 2759–2767 (2003).

    Article  CAS  PubMed  Google Scholar 

  76. West, G. M. et al. Ligand-dependent perturbation of the conformational ensemble for the GPCR β2 adrenergic receptor revealed by HDX. Structure 19, 1424–1432 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Horcajada, C., Guinovart, J. J., Fita, I. & Ferrer, J. C. Crystal structure of an archaeal glycogen synthase: Insights into oligomerization and substrate binding of eukaryotic glycogen synthases. J. Biol. Chem. 281, 2923–2931 (2006).

    Article  CAS  PubMed  Google Scholar 

  78. Thorsen, T. S., Matt, R., Weis, W. I. & Kobilka, B. K. Modified T4 lysozyme fusion proteins facilitate G protein-coupled receptor crystallogenesis. Structure 22, 1657–1664 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    Article  CAS  PubMed  Google Scholar 

  80. Shiroishi, M. et al. Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. Microb. Cell Fact. 11, 78 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Audet, M. et al. Small-scale approach for precrystallization screening in GPCR X-ray crystallography. Nat. Protoc. 15, 144–160 (2020).

    Article  CAS  PubMed  Google Scholar 

  82. Klenk, C., Ehrenmann, J., Schütz, M. & Plückthun, A. A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution. Sci. Rep. 6, 21294 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Popov, P. et al. Computational design of thermostabilizing point mutations for G protein-coupled receptors. eLife 7, e34729 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  84. Cherezov, V., Clogston, J., Papiz, M. Z. & Caffrey, M. Room to move: crystallizing membrane proteins in swollen lipidic mesophases. J. Mol. Biol. 357, 1605–1618 (2006).

    Article  CAS  PubMed  Google Scholar 

  85. Razinkov, I. et al. A new method for vitrifying samples for cryoEM. J. Struct. Biol. 195, 190–198 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Arnold, S. A. et al. Miniaturizing EM sample preparation: opportunities, challenges, and “visual proteomics”. Proteomics 18, 1700176 (2018).

    Article  Google Scholar 

  87. Drew, D., Lerch, M., Kunji, E., Slotboom, D.-J. & de Gier, J.-W. Optimization of membrane protein overexpression and purification using GFP fusions. Nat. Methods 3, 303–313 (2006).

    Article  CAS  PubMed  Google Scholar 

  88. Waltenspühl, Y., Jeliazkov, J. R., Kummer, L. & Plückthun, A. Directed evolution for high functional production and stability of a challenging G protein-coupled receptor. Sci. Rep. 11, 8630 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank G. Meier for his help during transient transfection and expression of protein and would furthermore like to thank F. Zosel for critical reading of the manuscript. This work was supported by Schweizerischer Nationalfonds grant 31003A_153143 and 31003A_182334, and KTI grant 18022.1 PFLS-LS, all to A.P.

Author information

Authors and Affiliations

Authors

Contributions

J.S. conceptualized the project, devised and established the thermostabilization platform including the expression format and microscale purification technique, performed NK1R mutagenesis and thermostabilization, and designed and characterized NK1R crystallization constructs. J.E. devised and established the SLIC-based mutagenesis and construct generation platform and performed the PTH1R thermostabilization and crystallization construct screening. Y.W. performed the OTR thermostabilization and crystallization construct screening with help from J.S. The project was supervised by A.P. The manuscript was prepared by J.S., J.E., Y.W. and A.P. All authors contributed to the final editing and approved of the manuscript.

Corresponding author

Correspondence to Andreas Plückthun.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Vadim Cherezov, Isabel Moraes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Ehrenmann, J. et al. Nat. Struct. Mol. Biol. 25, 1086–1092 (2018): https://doi.org/10.1038/s41594-018-0151-4

Schöppe, J. et al. Nat. Commun. 10, 17 (2019): https://doi.org/10.1038/s41467-018-07939-8

Waltenspühl, Y. et al. Sci. Adv. 6, eabb5419 (2020): https://doi.org/10.1126/sciadv.abb5419

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schöppe, J., Ehrenmann, J., Waltenspühl, Y. et al. Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP. Nat Protoc 17, 698–726 (2022). https://doi.org/10.1038/s41596-021-00660-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00660-9

  • Springer Nature Limited

Navigation