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Membrane Protein Preparation for Serial Crystallography Using High-Viscosity Injectors: Rhodopsin as an Example

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Expression, Purification, and Structural Biology of Membrane Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2127))

Abstract

Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new technique allowing to merge diffraction data from thousands of injector-delivered crystals that are too tiny for classical single-crystal analysis even in cryogenic conditions. The application of serial crystallography for studying protein dynamics is mentioned.

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References

  1. Standfuss J, Spence J (2017) Serial crystallography at synchrotrons and X-ray lasers. IUCrJ 4(Pt 2):100–101. https://doi.org/10.1107/S2052252517001877

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Barty A, Caleman C, Aquila A, Timneanu N, Lomb L, White TA, Andreasson J, Arnlund D, Bajt S, Barends TR, Barthelmess M, Bogan MJ, Bostedt C, Bozek JD, Coffee R, Coppola N, Davidsson J, Deponte DP, Doak RB, Ekeberg T, Elser V, Epp SW, Erk B, Fleckenstein H, Foucar L, Fromme P, Graafsma H, Gumprecht L, Hajdu J, Hampton CY, Hartmann R, Hartmann A, Hauser G, Hirsemann H, Holl P, Hunter MS, Johansson L, Kassemeyer S, Kimmel N, Kirian RA, Liang M, Maia FR, Malmerberg E, Marchesini S, Martin AV, Nass K, Neutze R, Reich C, Rolles D, Rudek B, Rudenko A, Scott H, Schlichting I, Schulz J, Seibert MM, Shoeman RL, Sierra RG, Soltau H, Spence JC, Stellato F, Stern S, Struder L, Ullrich J, Wang X, Weidenspointner G, Weierstall U, Wunderer CB, Chapman HN (2012) Self-terminating diffraction gates femtosecond X-ray nanocrystallography measurements. Nat Photonics 6:35–40. https://doi.org/10.1038/nphoton.2011.297

    Article  CAS  PubMed  Google Scholar 

  3. White TA, Barty A, Stellato F, Holton JM, Kirian RA, Zatsepin NA, Chapman HN (2013) Crystallographic data processing for free-electron laser sources. Acta Crystallogr D Biol Crystallogr 69(Pt 7):1231–1240. https://doi.org/10.1107/S0907444913013620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Neutze R, Wouts R, van der Spoel D, Weckert E, Hajdu J (2000) Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406(6797):752–757. https://doi.org/10.1038/35021099

    Article  CAS  PubMed  Google Scholar 

  5. Cheng RKY, Abela R, Hennig M (2017) X-ray free electron laser: opportunities for drug discovery. Essays Biochem 61(5):529–542. https://doi.org/10.1042/EBC20170031

    Article  PubMed  Google Scholar 

  6. Stauch B, Cherezov V (2018) Serial femtosecond crystallography of G protein-coupled receptors. Annu Rev Biophys 47:377–397. https://doi.org/10.1146/annurev-biophys-070317-033239

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Johansson LC, Stauch B, Ishchenko A, Cherezov V (2017) A bright future for serial femtosecond crystallography with XFELs. Trends Biochem Sci 42(9):749–762. https://doi.org/10.1016/j.tibs.2017.06.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Landau EM, Rosenbusch JP (1996) Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc Natl Acad Sci U S A 93(25):14532–14535

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Weierstall U, James D, Wang C, White TA, Wang D, Liu W, Spence JC, Bruce Doak R, Nelson G, Fromme P, Fromme R, Grotjohann I, Kupitz C, Zatsepin NA, Liu H, Basu S, Wacker D, Han GW, Katritch V, Boutet S, Messerschmidt M, Williams GJ, Koglin JE, Marvin Seibert M, Klinker M, Gati C, Shoeman RL, Barty A, Chapman HN, Kirian RA, Beyerlein KR, Stevens RC, Li D, Shah ST, Howe N, Caffrey M, Cherezov V (2014) Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat Commun 5:3309. https://doi.org/10.1038/ncomms4309

    Article  CAS  PubMed  Google Scholar 

  10. Nogly P, Panneels V, Nelson G, Gati C, Kimura T, Milne C, Milathianaki D, Kubo M, Wu W, Conrad C, Coe J, Bean R, Zhao Y, Bath P, Dods R, Harimoorthy R, Beyerlein KR, Rheinberger J, James D, DePonte D, Li C, Sala L, Williams GJ, Hunter MS, Koglin JE, Berntsen P, Nango E, Iwata S, Chapman HN, Fromme P, Frank M, Abela R, Boutet S, Barty A, White TA, Weierstall U, Spence J, Neutze R, Schertler G, Standfuss J (2016) Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography. Nat Commun 7:12314. https://doi.org/10.1038/ncomms12314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nogly P, James D, Wang D, White TA, Zatsepin N, S A, Nelson G, Liu H, Johansson L, Heymann M, J K, M M, Wickstrand C, Wu W, B P, B P, Oberthuer D, Panneels V, Cherezov V, Chapman H, Schertler G, Neutze R, Spence J, Moraes I, Burghammer M, Standfuss J, Weierstall U (2015) Lipidic cubic phase serial millisecond crystallography using synchrotron radiation. IUCrJ 2:168–176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang CY, Olieric V, Ma P, Panepucci E, Diederichs K, Wang M, Caffrey M (2015) In meso in situ serial X-ray crystallography of soluble and membrane proteins. Acta Crystallogr D Biol Crystallogr 71(Pt 6):1238–1256. https://doi.org/10.1107/S1399004715005210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Huang CY, Olieric V, Howe N, Warshamanage R, Weinert T, Panepucci E, Vogeley L, Basu S, Diederichs K, Caffrey M, Wang M (2018) In situ serial crystallography for rapid de novo membrane protein structure determination. Commun Biol 1:124. https://doi.org/10.1038/s42003-018-0123-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Martiel I, Muller-Werkmeister HM, Cohen AE (2019) Strategies for sample delivery for femtosecond crystallography. Acta Crystallogr D Struct Biol 75(Pt 2):160–177. https://doi.org/10.1107/S2059798318017953

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Weinert T, Olieric N, Cheng R, Brunle S, James D, Ozerov D, Gashi D, Vera L, Marsh M, Jaeger K, Dworkowski F, Panepucci E, Basu S, Skopintsev P, Dore AS, Geng T, Cooke RM, Liang M, Prota AE, Panneels V, Nogly P, Ermler U, Schertler G, Hennig M, Steinmetz MO, Wang M, Standfuss J (2017) Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons. Nat Commun 8(1):542. https://doi.org/10.1038/s41467-017-00630-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Edwards PC, Li J, Burghammer M, McDowell JH, Villa C, Hargrave PA, Schertler GF (2004) Crystals of native and modified bovine rhodopsins and their heavy atom derivatives. J Mol Biol 343(5):1439–1450. https://doi.org/10.1016/j.jmb.2004.08.089.S0022-2836(04)01089-7[pii].

    Article  CAS  PubMed  Google Scholar 

  17. Li D, Boland C, Aragao D, Walsh K, Caffrey M (2012) Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography. J Vis Exp 67:e4001. https://doi.org/10.3791/4001

    Article  CAS  Google Scholar 

  18. White TA, Kirian RA, Martin AV, Aquila A, Nass K, Barty A, Chapman HN (2012) CrystFEL: a software suite for snapshot serial crystallography. J Appl Cryst 45:335–334

    Article  CAS  Google Scholar 

  19. White TA, Mariani V, Brehm W, Yefanov O, Barty A, Beyerlein KR, Chervinskii F, Galli L, Gati C, Nakane T, Tolstikova A, Yamashita K, Yoon CH, Diederichs K, Chapman HN (2016) Recent developments in CrystFEL. J Appl Cryst 49(Pt 2):680–689. https://doi.org/10.1107/S1600576716004751

    Article  CAS  Google Scholar 

  20. McPherson A, Gavira JA (2014) Introduction to protein crystallization. Acta Crystallogr F Struct Biol Commun 70:2–20. https://doi.org/10.1107/S2053230X13033141

    Article  CAS  PubMed  Google Scholar 

  21. Lin SW, Sakmar TP (1996) Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state. Biochemistry 35(34):11149–11159. https://doi.org/10.1021/bi960858u

    Article  CAS  PubMed  Google Scholar 

  22. Caffrey M, Cherezov V (2009) Crystallizing membrane proteins using lipidic mesophases. Nat Protoc 4(5):706–731. https://doi.org/10.1038/nprot.2009.31.nprot.2009.31[pii].

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ishchenko A, Cherezov V, Liu W (2016) Preparation and delivery of protein microcrystals in lipidic cubic phase for serial femtosecond crystallography. J Vis Exp 115. https://doi.org/10.3791/54463

  24. Jaeger K, Dworkowski F, Nogly P, Milne C, Wang M, Standfuss J (2016) Serial millisecond crystallography of membrane proteins. Adv Exp Med Biol 922:137–149. https://doi.org/10.1007/978-3-319-35072-1_10

    Article  CAS  PubMed  Google Scholar 

  25. James D, Weinert T, Skopintsev P, Furrer A, Gashi D, Tanaka T, Nango E, Nogly P, Standfuss J (2019) Improving high viscosity extrusion of microcrystals for time-resolved serial femtosecond crystallography at X-ray lasers. J Vis Exp 144(e59087). https://doi.org/10.3791/59087

  26. Caffrey M, Porter C (2010) Crystallizing membrane proteins for structure determination using lipidic mesophases. J Vis Exp 45. https://doi.org/10.3791/1712

  27. Mariani V, Morgan A, Yoon CH, Lane TJ, White TA, O’Grady C, Kuhn M, Aplin S, Koglin J, Barty A, Chapman HN (2016) OnDA: online data analysis and feedback for serial X-ray imaging. J Appl Cryst 49(Pt 3):1073–1080. https://doi.org/10.1107/S1600576716007469

    Article  CAS  Google Scholar 

  28. Nakane T, Joti Y, Tono K, Yabashi M, Nango E, Iwata S, Ishitani R, Nureki O (2016) Data processing pipeline for serial femtosecond crystallography at SACLA. J Appl Cryst 49(Pt 3):1035–1041. https://doi.org/10.1107/S1600576716005720

    Article  CAS  Google Scholar 

  29. Haupert LM, Simpson GJ (2011) Screening of protein crystallization trials by second order nonlinear optical imaging of chiral crystals (SONICC). Methods 55(4):379–386. https://doi.org/10.1016/j.ymeth.2011.11.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kissick DJ, Wanapun D, Simpson GJ (2011) Second-order nonlinear optical imaging of chiral crystals. Annu Rev Anal Chem 4:419–437. https://doi.org/10.1146/annurev.anchem.111808.073722

    Article  CAS  Google Scholar 

  31. Nogly P, Weinert T, James D, Carbajo S, Ozerov D, Furrer A, Gashi D, Borin V, Skopintsev P, Jaeger K, Nass K, Bath P, Bosman R, Koglin J, Seaberg M, Lane T, Kekilli D, Brunle S, Tanaka T, Wu W, Milne C, White T, Barty A, Weierstall U, Panneels V, Nango E, Iwata S, Hunter M, Schapiro I, Schertler G, Neutze R, Standfuss J (2018) Retinal isomerization in bacteriorhodopsin captured by a femtosecond X-ray laser. Science 361(6398):eaat0094. https://doi.org/10.1126/science.aat0094

    Article  CAS  PubMed  Google Scholar 

  32. Weinert T, Skopintsev P, James D, Dworkowski F, Panepucci E, Kekilli D, Furrer A, Brunle S, Mous S, Ozerov D, Nogly P, Wang M, Standfuss J (2019) Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography. Science 365(6448):61–65. https://doi.org/10.1126/science.aaw8634

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Acknowledgments

We thank Thomas Gruhl for helping in developing the present protocol for rhodopsin serial crystallography and providing some pictures. We thank Edwards Stuttfeld for the continuous support and advices to work with the SONICC imager, and Timm Maier for the access to the SONICC imager. We acknowledge the Paul Scherrer Institut, Villigen, Switzerland, for provision of synchrotron radiation beam time at beamline PXI of the SLS and the continuous support from Andrea Prota and Takashi Tomizaki. This work was supported by the NCCR Molecular Systems Engineering 2015–2017 and Swiss National Science Foundation grant 173335 (to Gebhard Schertler). We acknowledge Filip Pamula and Maximilian Wranik for critical reading of the manuscript.

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Authors and Affiliations

  1. Division of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland

    Tobias Weinert & Valérie Panneels

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  1. Tobias Weinert
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  2. Valérie Panneels
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Correspondence to Tobias Weinert or Valérie Panneels .

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Editors and Affiliations

  1. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Camilo Perez

  2. Biozentrum, University of Basel, Basel, Basel Stadt, Switzerland

    Timm Maier

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Weinert, T., Panneels, V. (2020). Membrane Protein Preparation for Serial Crystallography Using High-Viscosity Injectors: Rhodopsin as an Example. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_21

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  • DOI: https://doi.org/10.1007/978-1-0716-0373-4_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0372-7

  • Online ISBN: 978-1-0716-0373-4

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