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De novo protein crystal structure determination from X-ray free-electron laser data

Abstract

The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized crystals1,2,3,4 in a ‘diffraction-before-destruction’ approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures1,2,3,4,5. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography1,2,3,4,6, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration6,7, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins1,2,8.

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Figure 1: w = 0.5 section of the origin-removed, super-sharpened anomalous difference Patterson map of the SFX lysozyme gadolinium data, using 60,000 images.
Figure 2: Quality of the phases at the various stages of the phasing process.
Figure 3: Progression of the phasing process.
Figure 4: Data quality as a function of resolution and number of indexed patterns used to derive integrated intensities as shown by Rsplit.

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Accession codes

Accessions

Protein Data Bank

Data deposits

Structure factor amplitudes and anomalous differences have been deposited in the Protein Data Bank along with the refined structure with accession code 4N5R, and diffraction patterns of crystal hits will be deposited at http://cxidb.org/.

References

  1. Chapman, H. N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011)

    Article  ADS  CAS  Google Scholar 

  2. Johansson, L. C. et al. Lipidic phase membrane protein serial femtosecond crystallography. Nature Methods 9, 263–265 (2012)

    Article  CAS  Google Scholar 

  3. Boutet, S. et al. High-resolution protein structure determination by serial femtosecond crystallography. Science 337, 362–364 (2012)

    Article  ADS  CAS  Google Scholar 

  4. Redecke, L. et al. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser. Science 339, 227–230 (2013)

    Article  ADS  CAS  Google Scholar 

  5. Kern, J. et al. Room temperature femtosecond X-ray diffraction of photosystem II microcrystals. Proc. Natl Acad. Sci. USA 109, 9721–9726 (2012)

    Article  ADS  CAS  Google Scholar 

  6. Kirian, R. A. et al. Femtosecond protein nanocrystallography-data analysis methods. Opt. Express 18, 5713–5723 (2010)

    Article  ADS  Google Scholar 

  7. White, T. A. et al. CrystFEL: a software suite for snapshot serial crystallography. J. Appl. Cryst. 45, 335–341 (2012)

    Article  CAS  Google Scholar 

  8. Kern, J. et al. Simultaneous femtosecond X-ray spectroscopy and diffraction of photosystem II at room temperature. Science 340, 491–495 (2013)

    Article  ADS  CAS  Google Scholar 

  9. Owen, R. L., Rudino-Pinera, E. & Garman, E. F. Experimental determination of the radiation dose limit for cryocooled protein crystals. Proc. Natl Acad. Sci. USA 103, 4912–4917 (2006)

    Article  ADS  CAS  Google Scholar 

  10. Spence, J. C. H. et al. Phasing of coherent femtosecond X-ray diffraction from size-varying nanocrystals. Opt. Express 19, 2866–2873 (2011)

    Article  ADS  CAS  Google Scholar 

  11. Banumathi, S., Zwart, P. H., Ramagopal, U. A., Dauter, M. & Dauter, Z. Structural effects of radiation damage and its potential for phasing. Acta Crystallogr. D 60, 1085–1093 (2004)

    Article  Google Scholar 

  12. Ravelli, R. B. G., Leiros, H.-K. S., Pan, B., Caffrey, M. & McSweeney, S. Specific radiation damage can be used to solve macromolecular crystal structures. Structure 11, 217–224 (2003)

    Article  CAS  Google Scholar 

  13. Son, S.-K., Chapman, H. N. & Santra, R. Multiwavelength anomalous diffraction at high X-ray intensity. Phys. Rev. Lett. 107, 218102 (2011)

    Article  ADS  Google Scholar 

  14. Weierstall, U., Spence, J. C. H. & Doak, R. B. Injector for scattering measurements on fully solvated biospecies. Rev. Sci. Instrum. 83, 035108 (2012)

    Article  ADS  CAS  Google Scholar 

  15. Sierra, R. G. et al. Nanoflow electrospinning serial femtosecond crystallography. Acta Crystallogr. D 68, 1584–1587 (2012)

    Article  CAS  Google Scholar 

  16. Kirian, R. A. et al. Structure-factor analysis of femtosecond micro-diffraction patterns from protein nanocrystals. Acta Crystallogr. A 67, 131–140 (2011)

    Article  ADS  CAS  Google Scholar 

  17. Barends, T. R. M. et al. Anomalous signal from S atoms in protein crystallographic data from an X-ray free-electron laser. Acta Crystallogr. D 69, 838–842 (2013)

    Article  CAS  Google Scholar 

  18. Girard, E., Chantalat, L., Vicat, J. & Kahn, R. Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozyme. Acta Crystallogr. D 58, 1–9 (2002)

    Article  Google Scholar 

  19. Boutet, S. & Williams, G. J. The Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). New J. Phys. 12, 035024 (2010)

    Article  ADS  Google Scholar 

  20. Foucar, L. et al. CASS-CFEL-ASG software suite. Comput. Phys. Commun. 183, 2207–2213 (2012)

    Article  ADS  CAS  Google Scholar 

  21. Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

    Article  CAS  Google Scholar 

  22. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  Google Scholar 

  23. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  24. Cowtan, K. ‘dm': An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31,. 34–38 (1994)

    Google Scholar 

  25. Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 1171–1179 (2008)

    Article  CAS  Google Scholar 

  26. Diederichs, K. & Karplus, P. A. Improved R-factors for diffraction data analysis in macromolecular crystallography. Nature Struct. Biol. 4, 269–275 (1997)

    Article  CAS  Google Scholar 

  27. Weiss, M. S. Global indicators of X-ray data quality. J. Appl. Crystallogr. 34, 130–135 (2001)

    Article  CAS  Google Scholar 

  28. Weiss, M. S., Sicker, T. & Hilgenfeld, R. Soft X-rays, high redundancy, and proper scaling: a new procedure for automated protein structure determination via SAS. Structure 9, 771–777 (2001)

    Article  CAS  Google Scholar 

  29. Panjikar, S. & Tucker, P. A. Phasing possibilities using different wavelengths with a xenon derivative. J. Appl. Crystallogr. 35, 261–266 (2002)

    Article  CAS  Google Scholar 

  30. Amann, J. et al. Demonstration of self-seeding in a hard-X-ray free-electron laser. Nature Photon. 6, 693–698 (2012)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

Portions of this research were carried out at the Linac Coherent Light Source, a National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The CXI instrument was funded by the LCLS Ultrafast Science Instruments (LUSI) project funded by the US Department of Energy, Office of Basic Energy Sciences. We acknowledge support from the Max Planck Society and from the EU for an Incoming Scientist Award to R.B.D. We thank the staff at the LCLS for their support and are grateful to S. Pesch and R. van Gessel (Bracco Imaging Konstanz and Singen, Germany) for the gift of the sample of gadoteridol. We thank H. Zimmermann for suggestions, W. Kabsch for discussions and J. Wray for critically reading the manuscript. In addition, we acknowledge L. Hammon and C. Patty for laboratory support, and the MCC staff for the beam they provided. We are indebted to C. Roome and F. Koeck for computing support.

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Authors and Affiliations

Authors

Contributions

T.R.M.B. and I.S. conceived the research, I.S. prepared crystals, Sa.B., R.B.D. and R.L.S. performed sample injection. Sé.B., G.J.W., J.E.K. and M.M. performed data collection, T.R.M.B., L.F. and K.N. performed data processing and analysis. T.R.M.B. and I.S. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Thomas R. M. Barends or Ilme Schlichting.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Anomalous signal strength of the SFX data (blue lines) as well as the rotating anode data (red lines) as measured by Rano on intensities (solid lines).

The noise in the data is indicated in terms of Rsplit for the SFX data and Rp.i.m. for the rotating anode data (dashed lines).

Extended Data Figure 2 Expected anomalous signal strength for a SAD experiment on lysozyme.

Expected anomalous signal strength for a SAD experiment on lysozyme with 2 gadolinium atoms per protein molecule at 8.5 keV (top panel) and for a sulphur-SAD experiment on lysozyme with 10 sulphur atoms per protein molecule at 6.0 keV (bottom panel). In each case, an optimistic scenario with all anomalous scatterers ordered is shown (green line) as well as a pessimistic scenario in which 60% of the anomalous scatterers are ordered (blue line). This figure was prepared using the anomalous scattering web server at http://skuld.bmsc.washington.edu/scatter/AS_signal.html.

Extended Data Table 1 Data collection, phasing and refinement statistics

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Supplementary Information

This file contains Supplementary Methods and Supplementary References. (PDF 328 kb)

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Barends, T., Foucar, L., Botha, S. et al. De novo protein crystal structure determination from X-ray free-electron laser data. Nature 505, 244–247 (2014). https://doi.org/10.1038/nature12773

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