Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Nature Protocols volume 4, pages 619–637 (2009)Cite this article

Abstract

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Workflow for generating membrane protein crystals.
Figure 2: Detergent solubilization of membrane proteins.
Figure 3: Size-exclusion chromatography.
Figure 4: Example of grid screens.
Figure 5: Scoring membrane protein crystal trials.
Figure 6: Flowchart for crystal optimization.

Similar content being viewed by others

References

  1. Kendrew, J.C. et al. A three-dimensional model of the myoglobin molecule obtained by X-ray analysis. Nature 181, 662–666 (1958).

    CAS  PubMed  Google Scholar 

  2. Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel, H. Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3Å resolution. Nature 318, 618–624 (1985).

    CAS  PubMed  Google Scholar 

  3. Doyle, D.A. et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69–77 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Ostermeier, C. & Michel, H. Crystallization of membrane proteins. Curr. Opin. Struct. Biol. 7, 697–701 (1997).

    CAS  PubMed  Google Scholar 

  5. Opekarova, M. & Tanner, W. Specific lipid requirements of membrane proteins—a putative bottleneck in heterologous expression. Biochim. Biophys. Acta 1610, 11–22 (2003).

    CAS  PubMed  Google Scholar 

  6. Cregg, J.M., Cereghino, J.L., Shi, J. & Higgins, D.R. Recombinant protein expression in Pichia pastoris . Mol. Biotechnol. 16, 23–52 (2000).

    CAS  PubMed  Google Scholar 

  7. Cereghino, J.L. & Cregg, J.M. Heterologous protein expression in the methylotrophic yeast Pichia pastoris . FEMS Microbiol. Rev. 24, 45–66 (2000).

    CAS  PubMed  Google Scholar 

  8. Long, S.B., Campbell, E.B. & Mackinnon, R. Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005).

    CAS  PubMed  Google Scholar 

  9. Tornroth-Horsefield, S. et al. Structural mechanism of plant aquaporin gating. Nature 439, 688–694 (2006).

    PubMed  Google Scholar 

  10. Jidenko, M. et al. Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 102, 11687–11691 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Pedersen, B.P., Buch-Pedersen, M.J., Morth, J.P., Palmgren, M.G. & Nissen, P. Crystal structure of the plasma membrane proton pump. Nature 450, 1111–1114 (2007).

    CAS  PubMed  Google Scholar 

  12. Jasti, J., Furukawa, H., Gonzales, E.B. & Gouaux, E. Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH. Nature 449, 316–323 (2007).

    CAS  PubMed  Google Scholar 

  13. Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Reeves, P.J., Kim, J.M. & Khorana, H.G. Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc. Natl. Acad. Sci. USA 99, 13413–13418 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Gonen, T. et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438, 633–638 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Oxenoid, K., Kim, H.J., Jacob, J., Sonnichsen, F.D. & Sanders, C.R. NMR assignments for a helical 40 kDa membrane protein. J. Am. Chem. Soc. 126, 5048–5049 (2004).

    CAS  PubMed  Google Scholar 

  17. Opella, S.J. & Marassi, F.M. Structure determination of membrane proteins by NMR spectroscopy. Chem. Rev. 104, 3587–3606 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Howell, S.C., Mesleh, M.F. & Opella, S.J. NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system. Biochemistry 44, 5196–5206 (2005).

    CAS  PubMed  Google Scholar 

  19. Tian, C. et al. Membrane protein preparation for TROSY NMR screening. Methods Enzymol. 394, 321–334 (2005).

    CAS  PubMed  Google Scholar 

  20. Rigaud, J.L. & Levy, D. Reconstitution of membrane proteins into liposomes. Methods Enzymol. 372, 65–86 (2003).

    CAS  PubMed  Google Scholar 

  21. Morera, F.J., Vargas, G., Gonzalez, C., Rosenmann, E. & Latorre, R. Ion-channel reconstitution. Methods Mol. Biol. 400, 571–585 (2007).

    CAS  PubMed  Google Scholar 

  22. Andreoli, T.E. Planar lipid bilayer membranes. Methods Enzymol. 32, 513–539 (1974).

    CAS  PubMed  Google Scholar 

  23. Bylund, D.B. & Toews, M.L. Radioligand binding methods: practical guide and tips. Am. J. Physiol. 265, L421–L429 (1993).

    CAS  PubMed  Google Scholar 

  24. Bylund, D.B., Deupree, J.D. & Toews, M.L. Radioligand-binding methods for membrane preparations and intact cells. Methods Mol. Biol. 259, 1–28 (2004).

    CAS  PubMed  Google Scholar 

  25. Lebowitz, J., Lewis, M.S. & Schuck, P. Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci. 11, 2067–2079 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Hong, X., Weng, Y.X. & Li, M. Determination of the topological shape of integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria in the detergent solution by small-angle X-ray scattering. Biophys. J. 86, 1082–1088 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Lipfert, J. & Doniach, S. Small-angle X-ray scattering from RNA, proteins, and protein complexes. Annu. Rev. Biophys. Biomol. Struct. 36, 307–327 (2007).

    CAS  PubMed  Google Scholar 

  28. Putnam, C.D., Hammel, M., Hura, G.L. & Tainer, J.A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q Rev. Biophys. 40, 191–285 (2007).

    CAS  PubMed  Google Scholar 

  29. Folta-Stogniew, E. & Williams, K.R. Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J. Biomol. Techniques 10, 51–63 (1999).

    CAS  Google Scholar 

  30. Swords, N.A. & Wallace, B.A. Circular-dichroism analyses of membrane proteins: examination of environmental effects on bacteriorhodopsin spectra. Biochem. J. 289 (Part 1): 215–219 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Wallace, B.A., Lees, J.G., Orry, A.J., Lobley, A. & Janes, R.W. Analyses of circular dichroism spectra of membrane proteins. Protein Sci. 12, 875–884 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Fu, D., Libson, A. & Stroud, R. The structure of GlpF, a glycerol conducting channel. Novartis Found Symp. 245, 51–61; discussion 61–65, 165–168 (2002).

    CAS  PubMed  Google Scholar 

  33. Savage, D.F., Egea, P.F., Robles-Colmenares, Y., O'Connell, J.D. III & Stroud, R.M. Architecture and selectivity in aquaporins: 2.5 a X-ray structure of aquaporin Z. PLoS Biol. 1, E72 (2003).

    PubMed  PubMed Central  Google Scholar 

  34. Harries, W.E., Akhavan, D., Miercke, L.J., Khademi, S. & Stroud, R.M. The channel architecture of aquaporin 0 at a 2.2-A resolution. Proc. Natl. Acad. Sci. USA 101, 14045–14050 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Khademi, S. et al. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305, 1587–1594 (2004).

    CAS  PubMed  Google Scholar 

  36. Lee, J.K. et al. Structural basis for conductance by the archaeal aquaporin AqpM at 1.68 A. Proc. Natl. Acad. Sci. USA 102, 18932–18937 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Gruswitz, F., O'Connell, J. III & Stroud, R.M. Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96 A. Proc. Natl. Acad. Sci. USA 104, 42–47 (2007).

    CAS  PubMed  Google Scholar 

  38. Newby, Z.E. et al. Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum. Nat. Struct. Mol. Biol. 15, 619–625 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Caffrey, M. Membrane protein crystallization. J. Struct. Biol. 142, 108–132 (2003).

    CAS  PubMed  Google Scholar 

  40. Garavito, R.M., Markovic-Housley, Z. & Jenkins, J.A. The growth and characterization of membrane protein crystals. J. Cryst. Growth 76, 701–709 (1986).

    CAS  Google Scholar 

  41. Garavito, R.M., Picot, D. & Loll, P.J. Strategies for crystallizing membrane proteins. J. Bioenerg. Biomembr. 28, 13–27 (1996).

    CAS  PubMed  Google Scholar 

  42. Iwata, S. Methods and Results in Crystallization of Membrane Proteins 355 (International University Line, La Jolla, CA, 2003).

    Google Scholar 

  43. Michel, H. Crystallization of Membrane Proteins (CRC Press, Boca Raton, FL, 1991).

    Google Scholar 

  44. Wiener, M.C. A pedestrian guide to membrane protein crystallization. Methods 34, 364–372 (2004).

    CAS  PubMed  Google Scholar 

  45. Hemdan, E.S. & Porath, J. Development of immobilized metal affinity chromatography II. Interaction of amino acids with immobilized nickel iminodiacetate. J. Chromatogr. 323, 255–264 (1985).

    CAS  Google Scholar 

  46. Hemdan, E.S. & Porath, J. Development of immobilized metal affinity chromatography III. Interaction of oligopeptides with immobilized nickel iminodiacetate. J. Chromatogr. 323, 255–264 (1985).

    CAS  Google Scholar 

  47. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89 (1990).

    CAS  PubMed  Google Scholar 

  48. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Van den Berg, B. et al. X-ray structure of a protein-conducting channel. Nature 427, 36–44 (2004).

    CAS  PubMed  Google Scholar 

  50. Junge, F. et al. Large-scale production of functional membrane proteins. Cell Mol. Life Sci. (2008).

  51. Hilf, R.J. & Dutzler, R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature 452, 375–379 (2008).

    CAS  PubMed  Google Scholar 

  52. Roosild, T.P. et al. NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science 307, 1317–1321 (2005).

    CAS  PubMed  Google Scholar 

  53. Makrides, S.C. Strategies for achieving high-level expression of genes in Escherichia coli . Microbiol. Rev. 60, 512–538 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Ferguson, A.D. et al. Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317, 510–512 (2007).

    CAS  PubMed  Google Scholar 

  55. Miroux, B. & Walker, J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

    CAS  PubMed  Google Scholar 

  56. Hannig, G. & Makrides, S.C. Strategies for optimizing heterologous protein expression in Escherichia coli . Trends Biotechnol. 16, 54–60 (1998).

    CAS  PubMed  Google Scholar 

  57. Yeliseev, A.A., Wong, K.K., Soubias, O. & Gawrisch, K. Expression of human peripheral cannabinoid receptor for structural studies. Protein Sci. 14, 2638–2653 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Raman, P., Cherezov, V. & Caffrey, M. The membrane protein data bank. Cell Mol. Life Sci. 63, 36–51 (2006).

    CAS  PubMed  Google Scholar 

  59. Newstead, S., Ferrandon, S. & Iwata, S. Rationalizing alpha-helical membrane protein crystallization. Protein Sci. 17, 466–472 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Garavito, R.M. & Ferguson-Miller, S. Detergents as tools in membrane biochemistry. J. Biol. Chem. 276, 32403–32406 (2001).

    CAS  PubMed  Google Scholar 

  61. Chiu, M.L., Tsang, C., Grihalde, N. & MacWilliams, M.P. Over-expression, solubilization, and purification of G protein-coupled receptors for structural biology. Comb. Chem. High Throughput Screen 11, 439–462 (2008).

    CAS  PubMed  Google Scholar 

  62. Faham, S. & Bowie, J.U. Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biol. 316, 1–6 (2002).

    CAS  PubMed  Google Scholar 

  63. Weckstrom, K. Aqueous micellar systems in membrane protein crystallization. Partial miscibility of a nonionic surfactant in the presence of salt or polyethylene glycol. FEBS Lett. 192, 220–224 (1985).

    CAS  PubMed  Google Scholar 

  64. Mohanty, A.K., Simmons, C.R. & Wiener, M.C. Inhibition of tobacco etch virus protease activity by detergents. Protein Expr. Purif. 27, 109–114 (2003).

    CAS  PubMed  Google Scholar 

  65. Potschka, M. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Anal. Biochem. 162, 47–64 (1987).

    CAS  PubMed  Google Scholar 

  66. Barth, H.G., Boyes, B.E. & Jackson, C. Size exclusion chromatography. Anal. Chem. 66, 595R–620R (1994).

    CAS  PubMed  Google Scholar 

  67. Ricker, R.D. & Sandoval, L.A. Fast, reproducible size-exclusion chromatography of biological macromolecules. J. Chromatogr. A 743, 43–50 (1996).

    CAS  PubMed  Google Scholar 

  68. Trathnigg, B. Size-exclusion chromatography of polymers. in Encyclopedia of Analytical Chemistry (ed. Meyers, R.A.) 8008–8034 (John Wiley & Sons Ltd, Chichester, 2000).

    Google Scholar 

  69. Strop, P. & Brunger, A.T. Refractive index-based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci. 14, 2207–2211 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Bergfors, T.M. Protein Crystallization 306 (International University Line, La Jolla, CA, 1999).

    Google Scholar 

  71. Walter, T.S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. I. Protocol design and validation. J. Appl. Cryst. 36, 308–314 (2003).

    CAS  Google Scholar 

  72. McPherson, A. & Cudney, B. Searching for silver bullets: an alternative strategy for crystallizing macromolecules. J. Struct. Biol. 156, 387–406 (2006).

    CAS  PubMed  Google Scholar 

  73. Guan, L., Smirnova, I.N., Verner, G., Nagamori, S. & Kaback, H.R. Manipulating phospholipids for crystallization of a membrane transport protein. Proc. Natl. Acad. Sci. USA 103, 1723–1726 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. McFerrin, M.B. & Snell, E.H. The development and applicatioin of a method to quantify the quality of cryoprotectant solutions using standard area-detector X-ray images. J. Appl. Cryst. 35, 538–545 (2002).

    CAS  Google Scholar 

  75. Heras, B. & Martin, J.L. Post-crystallization treatments for improving diffraction quality of protein crystals. Acta Crystallogr. D Biol. Crystallogr. 61, 1173–1180 (2005).

    PubMed  Google Scholar 

  76. Cohen, S.L. & Chait, B.T. Mass spectrometry as a tool for protein crystallography. Annu. Rev. Biophys. Biomol. Struct. 30, 67–85 (2001).

    CAS  PubMed  Google Scholar 

  77. Hunte, C. & Michel, H. Crystallisation of membrane proteins mediated by antibody fragments. Curr. Opin. Struct. Biol. 12, 503–508 (2002).

    CAS  PubMed  Google Scholar 

  78. Jiang, Y. et al. X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41 (2003).

    CAS  PubMed  Google Scholar 

  79. Landau, E.M. & Rosenbusch, J.P. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl. Acad. Sci. USA 93, 14532–14535 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Nollert, P. Lipidic cubic phases as matrices for membrane protein crystallization. Methods 34, 348–353 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Ng, J.D., Gavira, J.A. & Garcia-Ruiz, J.M. Protein crystallization by capillary counterdiffusion for applied crystallographic structure determination. J. Struct. Biol. 142, 218–231 (2003).

    CAS  PubMed  Google Scholar 

  82. Hansen, C.L., Skordalakes, E., Berger, J.M. & Quake, S.R. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc. Natl. Acad. Sci. USA 99, 16531–16536 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Hansen, C. & Quake, S.R. Microfluidics in structural biology: smaller, faster em leader better. Curr. Opin. Struct. Biol. 13, 538–544 (2003).

    CAS  PubMed  Google Scholar 

  84. Hansen, C.L., Classen, S., Berger, J.M. & Quake, S.R. A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination. J. Am. Chem. Soc. 128, 3142–3143 (2006).

    CAS  PubMed  Google Scholar 

  85. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, New York, USA, 1989).

    Google Scholar 

  86. Savage, D.F., Anderson, C.L., Robles-Colmenares, Y., Newby, Z.E. & Stroud, R.M. Cell-free complements in vivo expression of the E. coli membrane proteome. Protein Sci. 16, 966–976 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. McPherson, A. Current approaches to macromolecular crystallization. Eur. J. Biochem. 189, 1–23 (1990).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Research was supported by NIH grant RO1 GM24485 to R.M.S., the NIH Roadmap center grant P50 GM073210 and the Specialized Center grant of the Protein Structure Initiative U54 GM074929-01. We thank Rebecca Robbins in the Stroud laboratory and Ryan R Atkinson for helpful discussions and help in preparing the manuscript. We also thank the reviewers and editors for their helpful suggestions.

Author information

Author notes

  1. David F Savage

    Present address: Present address: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California in San Francisco, 600 16th Street, San Francisco, 94158-2517, California, USA

    Zachary E R Newby, Joseph D O'Connell III, Franz Gruswitz, Franklin A Hays, William E C Harries, Ian M Harwood, Joseph D Ho, John K Lee, David F Savage, Larry J W Miercke & Robert M Stroud

Authors
  1. Zachary E R Newby
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph D O'Connell III
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Franz Gruswitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Franklin A Hays
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William E C Harries
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ian M Harwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joseph D Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. John K Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David F Savage
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Larry J W Miercke
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Robert M Stroud
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert M Stroud.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Newby, Z., O'Connell, J., Gruswitz, F. et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat Protoc 4, 619–637 (2009). https://doi.org/10.1038/nprot.2009.27

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.27

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing