Abstract
We have recently established a procedure for serial femtosecond crystallography (SFX) in lipidic cubic phase (LCP) for protein structure determination at X-ray free-electron lasers (XFELs). LCP-SFX uses the gel-like LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room-temperature high-resolution data with minimal effects of radiation damage from sub-10-μm crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared with SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2–3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3–5 d, including the time required for the crystals to grow.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Neutze, R., Wouts, R., van der Spoel, D., Weckert, E. & Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752–757 (2000).
Chapman, H.N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011).
Spence, J.C., Weierstall, U. & Chapman, H.N. X-ray lasers for structural and dynamic biology. Rep. Prog. Phys. 75, 102601 (2012).
White, T.A. et al. Crystallographic data processing for free-electron laser sources. Acta Crystallogr. D Biol. Crystallogr. 69, 1231–1240 (2013).
DePonte, D.P. et al. Gas dynamic virtual nozzle for generation of microscopic droplet streams. J. Phys. D Appl. Phys. 41, 195505 (2008).
Lomb, L. et al. An anti-settling sample delivery instrument for serial femtosecond crystallography. J. Appl. Cryst. 45, 674–678 (2012).
Johansson, L.C. et al. Lipidic phase membrane protein serial femtosecond crystallography. Nat. Methods 9, 263–265 (2012).
Boutet, S. et al. High-resolution protein structure determination by serial femtosecond crystallography. Science 337, 362–364 (2012).
Redecke, L. et al. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339, 227–230 (2013).
Johansson, L.C. et al. Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography. Nat. Commun. 4, 2911 (2013).
Kern, J. et al. Room temperature femtosecond X-ray diffraction of photosystem II microcrystals. Proc. Natl. Acad. Sci. USA 109, 9721–9726 (2012).
Sierra, R.G. et al. Nanoflow electrospinning serial femtosecond crystallography. Acta Crystallogr. D Biol. Crystallogr. 68, 1584–1587 (2012).
Weierstall, U. et al. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat. Commun. 5, 3309 (2014).
Larsson, K. Cubic lipid-water phases: structures and biomembrane aspects. J. Phys. Chem. 93, 7304–7314 (1989).
Cherezov, V. Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin. Struct. Biol. 21, 559–566 (2011).
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).
Liu, W. et al. Serial femtosecond crystallography of G protein-coupled receptors. Science 342, 1521–1524 (2013).
Zarrine-Afsar, A. et al. Crystallography on a chip. Acta Crystallogr. D Biol. Crystallogr. 68, 321–323 (2012).
Frank, M. et al. Femtosecond X-ray diffraction from two-dimensional protein crystals. IUCrJ 1, 95–100 (2014).
Gati, C. et al. Serial crystallography on in vivo-grown microcrystals using synchrotron radiation. IUCrJ 1, 87–94 (2014).
Kimura, T. et al. Imaging live cell in micro-liquid enclosure by X-ray laser diffraction. Nat. Commun. 5, 3052 (2014).
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).
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).
Liu, W. & Cherezov, V. Crystallization of membrane proteins in lipidic mesophases. J. Vis. Exp. 49, e2501 (2011).
Li, D., Boland, C., Walsh, K. & Caffrey, M. Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases. J. Vis. Exp. 67, e4000 (2012).
Kulkarni, C.V., Wachter, W., Iglesias-Salto, G., Engelskirchen, S. & Ahualli, S. Monoolein: a magic lipid? Phys. Chem. Chem. Phys. 13, 3004–3021 (2011).
Li, D., Shah, S.T. & Caffrey, M. Host lipid and temperature as important screening variables for crystallizing integral membrane proteins in lipidic mesophases. Trials with diacylglycerol kinase. Cryst. Growth Des. 13, 2846–2857 (2013).
Li, D. et al. Crystallizing membrane proteins in the lipidic mesophase. Experience with human prostaglandin E2 synthase 1 and an evolving strategy. Cryst. Growth Des. 14, 2034–2047 (2014).
Cherezov, V., Clogston, J., Misquitta, Y., Abdel-Gawad, W. & Caffrey, M. Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase. Biophys. J. 83, 3393–3407 (2002).
Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).
Qiu, H. & Caffrey, M. Lyotropic and thermotropic phase behavior of hydrated monoacylglycerols: structure characterization of monovaccenin. J. Phys. Chem. B 102, 4819–4829 (1998).
Qiu, H. & Caffrey, M. The phase diagram of the monoolein/water system: metastability and equilibrium aspects. Biomaterials 21, 223–234 (2000).
Misquitta, L.V. et al. Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 12, 2113–2124 (2004).
Misquitta, Y. et al. Rational design of lipid for membrane protein crystallization. J. Struct. Biol. 148, 169–175 (2004).
Cherezov, V., Fersi, H. & Caffrey, M. Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins. Biophys. J. 81, 225–242 (2001).
Joseph, J.S. et al. Characterization of lipid matrices for membrane protein crystallization by high-throughput small angle X-ray scattering. Methods 55, 342–349 (2011).
van 't Hag, L. et al. In meso crystallization: compatibility of different lipid bicontinuous cubic mesophases with the cubic crystallization screen in aqueous solution. Cryst. Growth Des. 14, 1771–1781 (2014).
Landau, E.M., Rummel, G., Cowan-Jacob, S.W. & Rosenbusch, J.P. Crystallization of a polar protein and small molecules from the aqueous compartment of lipidic cubic phases. J. Phys. Chem. B 101, 1935–1937 (1997).
Caffrey, M. A lipid's eye view of membrane protein crystallization in mesophases. Curr. Opin. Struct. Biol. 10, 486–497 (2000).
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).
Wadsten, P. et al. Lipidic sponge phase crystallization of membrane proteins. J. Mol. Biol. 364, 44–53 (2006).
Owen, R.L. et al. Outrunning free radicals in room-temperature macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 68, 810–818 (2012).
Wacker, D. et al. Structural features for functional selectivity at serotonin receptors. Science 340, 615–619 (2013).
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).
Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).
Liu, W. Membrane Protein Crystallization in the Lipidic Cubic Phase: Testing Hypotheses Relating to Reconstitution PhD thesis, (The Ohio State University, 2007).
Acknowledgements
This work was supported by the US National Institutes of Health grant nos. P50 GM073197 and U54 GM094618. We thank K. Kadyshevskaya for assistance with figure preparation, L. Johansson for comments and A. Walker for assistance with manuscript preparation.
Author information
Authors and Affiliations
Contributions
W.L. and A.I. worked out the protocols and wrote the initial draft, and V.C. developed the concept and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Liu, W., Ishchenko, A. & Cherezov, V. Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography. Nat Protoc 9, 2123–2134 (2014). https://doi.org/10.1038/nprot.2014.141
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2014.141
This article is cited by
-
Growing and making nano- and microcrystals
Nature Protocols (2023)
-
Structure-based drug discovery of a corticotropin-releasing hormone receptor 1 antagonist using an X-ray free-electron laser
Experimental & Molecular Medicine (2023)
-
Beef tallow injection matrix for serial crystallography
Scientific Reports (2022)
-
MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography
Nature Communications (2021)
-
Illumination guidelines for ultrafast pump–probe experiments by serial femtosecond crystallography
Nature Methods (2020)
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.