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Serial femtosecond crystallography

Abstract

With the advent of X-ray free-electron lasers (XFELs), new, high-throughput serial crystallography techniques for macromolecular structure determination have emerged. Serial femtosecond crystallography (SFX) and related methods provide possibilities beyond canonical, single-crystal rotation crystallography by mitigating radiation damage and allowing time-resolved studies with unprecedented temporal resolution. This Primer aims to assist structural biology groups with little or no experience in serial crystallography in planning and carrying out a successful SFX experiment. It discusses the background of serial crystallography and its possibilities. Microcrystal growth and characterization methods are discussed, alongside techniques for sample delivery and data processing. Moreover, it gives practical tips for preparing an experiment, what to consider and do during a beam time and how to conduct the final data analysis. Finally, the Primer looks at various applications of SFX, including structure determination of membrane proteins, investigation of radiation damage-prone systems and time-resolved studies.

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Fig. 1: Differences in experimental crystallography set-ups.
Fig. 2: SFX sample delivery methods.
Fig. 3: Distribution of sample delivery techniques used for published serial femtosecond crystallography experiments resulting in Protein Data Bank codes (data up to December 2021).
Fig. 4: Integration of intensities in rotation and serial crystallography.

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Acknowledgements

The authors thank the Heidelberg FEL group and the instrument scientists at the various XFEL facilities for numerous stimulating discussions over the years. This work was funded by National Institutes of Health grant R35 GM127086 and the Max Planck Society.

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Authors and Affiliations

Authors

Contributions

Introduction (T.R.M.B., B.S., V.C. and I.S.); Experimentation (T.R.M.B., B.S., V.C. and I.S.); Results (T.R.M.B., B.S., V.C. and I.S.); Applications (T.R.M.B., B.S., V.C. and I.S.); Reproducibility and data deposition (T.R.M.B., B.S., V.C. and I.S.); Limitations and optimizations (T.R.M.B., B.S., V.C. and I.S.); Outlook (T.R.M.B., B.S., V.C. and I.S.); Overview of the Primer (T.R.M.B., B.S., V.C. and I.S.).

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Correspondence to Ilme Schlichting.

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Nature Reviews Methods Primers thanks Jan Kern, who co-reviewed with Phillip Simon; Michael Thompson; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Alvra at SwissFEL: https://www.psi.ch/en/swissfel/alvra

BL2 and BL3 at SACLA: http://xfel.riken.jp/eng/users/bml04-11.html

CXI at LCLS: https://lcls.slac.stanford.edu/instruments/cxi/specifications

CXIDB: https://cxidb.org/

IRRMC: https://proteindiffraction.org/

MFX at LCLS: https://lcls.slac.stanford.edu/instruments/mfx/specifications

NCI SFX at PAL-XFEL: https://pal.postech.ac.kr/paleng/Menu.pal?method=menuView&pageMode=paleng&top=7&sub=2&sub2=2&sub3=4

SPB/SFX at EuXFEL: https://www.xfel.eu/facility/instruments/spb_sfx/index_eng.html

Supplementary information

Glossary

Lipidic cubic phase

A gel-like membrane-mimetic mesophase suitable for stabilization and crystallization of membrane proteins in a lipidic environment.

X-ray free-electron lasers

Radiation sources based on a linear electron accelerator and a very long undulator, a periodic array of magnets. Electrons travel through the undulator at relativistic speed and experience an alternating magnetic field, causing them to emit X-ray radiation. This emitted radiation acts back on the electrons, causing some to accelerate and others to decelerate, grouping them into microbunches spaced at the wavelength of the radiation. Because of this, the electrons emit in-phase, resulting in extremely intense pulses of coherent radiation. This process is known as self-amplified spontaneous emission.

Photoionization

The near-instantaneous emission of an electron, typically from an inner shell, caused by the absorption of a photon.

Auger electrons

An electron emitted from an atom following the earlier loss of an inner-shell electron due to, for example, photoionization. The vacancy caused by the loss of the first electron is filled by a higher-shell electron, and the energy released by this is transferred to a less tightly bound electron, which is then emitted as an Auger electron.

Repetition rate

The number of X-ray pulses per second produced by the laser. The rate varies from 10 Hz to 120 Hz for early X-ray free-electron lasers (XFELs) to several megahertz for XFELs using superconducting accelerators.

Fixed target

A crystal delivery method, in which crystals are grown inside a chip or deposited on a solid support, typically a chip. The solid support is rastered in the X-ray beam using fast XY stages.

Friedel mates

Pairs of reflections Fh,k,l and Fh,−k,−l that are not related by space group point symmetry have the same magnitude unless anomalous signal is present. Highly precise quantification of intensity differences facilitates certain techniques for experimental phasing.

Gas dynamic virtual nozzles

A device for jet-type crystal delivery that consists of an inner nozzle, through which a crystal suspension is pumped, surrounded by an outer nozzle through which a sheath gas is delivered. A constriction at the end of the outer nozzle causes the gas stream to narrow and accelerate. This virtual nozzle focuses the liquid stream into a narrow jet.

Hit rate

The fraction of images containing a diffraction pattern. In serial femtosecond crystallography, crystal delivery is typically not synchronized with the X-ray pulses and therefore, not every exposure results in a diffraction pattern.

Injector

A device used to introduce crystals into the X-ray beam in a thin liquid jet, gas-dynamic virtual nozzle injector, or viscous stream, high-viscosity extrusion injector, for serial data collection.

High-viscosity extrusion

(HVE). A special device for the delivery of crystals grown inside viscous media such as lipidic cubic phase or embedded into a viscous matrix. The HVE injector consists of a reservoir filled with viscous matrix containing crystals, a hydraulic or mechanical plunger and a narrow capillary through which the viscous medium is streamed. The stream is supported by a sheath gas, typically nitrogen or helium, to keep it straight and avoid curling back and sticking to the nozzle.

Drop-on-demand

A sample injection method in which a piezoelectric device generates small droplets of a crystal suspension synchronously with the arrival of the X-ray pulses.

Drop-on-tape

A sample injection method in which the droplets are deposited on a tape that moves the droplets through the beam as on a conveyor belt.

R split

A special R factor used for the assessment of the precision of serial data, calculated by randomly splitting the available images into two halves (A and B), integrating both to prepare two half datasets and calculating the R factor between them, corrected for the reduction in multiplicity.

CC1/2

A measure of the precision of X-ray crystallography data that can be applied to serial crystallography, calculated for serial data by randomly dividing the available images into two subsets, each of which is subjected to Monte-Carlo integration to obtain two sets of integrated intensities. The Pearson correlation between the two is then calculated.

Omit maps

An electron density map calculated from observed intensities and phases from a model that excludes certain atoms, or groups of atoms, with the objective of removing the contribution of these atoms to minimize model phase bias.

Indexing rate

The fraction of indexable diffraction patterns of all hits. Typically, in serial femtosecond crystallography not every collected diffraction pattern can be indexed, often for lacking sufficient suitable diffraction spots.

Extrapolated structure factors

Estimated structure factor amplitudes corresponding to 100% activated or ligated state occupancy, calculated by linear extrapolation of structure factore amplitudes measured from crystals with less than full occupancy.

Pump–probe

A method for collecting time-resolved serial femtosecond crystallography data, in which a laser flash pump is used to trigger a reaction and then its structure is probed after a specified time delay by the X-ray free-electron laser beam. The wavelength range of the pump spans the X-ray region to the terahertz region.

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Barends, T.R.M., Stauch, B., Cherezov, V. et al. Serial femtosecond crystallography. Nat Rev Methods Primers 2, 59 (2022). https://doi.org/10.1038/s43586-022-00141-7

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