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


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


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


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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



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

Extended Data Table 1 Data collection, phasing and refinement statistics

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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).

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