Serial femtosecond crystallography opens new avenues for Structural Biology

Abstract

Free electron lasers (FELs) provide X-ray pulses in the femtosecond time domain with up to 1012 higher photon flux than synchrotrons and open new avenues for the determination of difficult to crystallize proteins, like large complexes and human membrane proteins. While the X-ray pulses are so strong that they destroy any solid material, the crystals diffract before they are destroyed. The most successful application of FELs for biology has been the method of serial femtosecond crystallography (SFX) where nano or microcrystals are delivered to the FEL beam in a stream of their mother liquid at room temperature, which ensures the replenishment of the sample before the next X-ray pulse arrives. New injector technology allows also for the delivery of crystal in lipidic cubic phases or agarose, which reduces the sample amounts for an SFX data set by two orders of magnitude. Time-resolved SFX also allows for analysis of the dynamics of biomolecules, the proof of principle being recently shown for light-induced reactions in photosystem II and photoactive yellow protein. An SFX data sets consist of thousands of single crystal snapshots in random orientations, which can be analyzed now “on the fly” by data analysis programs specifically developed for SFX, but de-novo phasing is still a challenge, that might be overcome by two-color experiments or phasing by shape transforms.

Applications of X-ray Powder Diffraction in Protein Crystallography and Drug Screening

“…Furthermore, introduction of the free-electron X-ray laser (XFEL) to structure and dynamics in biology have the potential to prevent the effects of radiation damage [184]. XFELs provide femtosecond pulses with up to 10 12 higher photon flux than synchrotrons [185], allowing both structure determination and time-resolved studies of submicrometer crystals that are small XRPD measurements, by delivering them to the XFEL beam in a stream of their mother liquid at room temperature [186,187]. The speed and brightness offered by XFELs are crucial for certain types of experiments, and pulses are so short that data can be collected avoiding the effects of radiation damage [188].…”

“…This approach has been enormously powerful but is limited by the fact that the molecule or complex of interest must be crystallized, which is not always possible [191]. When macromolecules and complexes prove hard to crystallize or to be produced in the sufficient concentration to even attempt crystallization trials, 3D electron microscopy is a potential alternative to X-ray crystallography that is quickly gaining popularity among structural biologists [185]. In the electron microscope method, aiming to endure the high vacuum and minimize visible effects of radiation damage and thus highly affecting biological studies, samples can be then studied in a frozen hydrated state after vitrification (cryo-EM).…”

Fixed-Target Serial Crystallography at Structural Biology Center

“…SSX can capture structural changes, including binding of metals, molecules, and cofactors, and recording chemical transformation, including enzymatic catalysis, in a time-dependent manner (manuscript in preparation). Structures measured at different time points can help visualize structural and chemical changes with time scales spanning sixteen orders of magnitude, from femtoseconds 2 to minutes 24 . While it still takes thousands of crystals to measure each time point, the advancements in modern light sources, delivery methods, detectors, and analysis pipelines have converged to make atomic-resolution structural dynamics an exciting new investigative option.…”

“…SSX followed and refined serial crystallography experiments 1 performed at x-ray Free-Electron Lasers (XFELs). The need arose because the powerful pulses from XFELs destroy each crystal after a single exposure, and thousands of crystals must be exposed to capture enough diffraction reflections to solve and refine the structure 2,3 . Improvements in technology since the introduction of serial crystallography have made SSX much more accessible and routine [4][5][6][7][8][9][10][11] .…”

New perspectives in macromolecular powder diffraction using single-photon-counting strip detectors: high-resolution structure of the pharmaceutical peptide octreotide

“…Despite the wide range of spectroscopic and scattering 'in-solution' techniques, as well as 3D electron microscopy which is quickly gaining ground among structural biologists (Yip et al, 2020;Nakane et al, 2020), X-ray crystallography is still the primary technique for determining macromolecular structures at atomic resolution (Egelman, 2016), since it is not limited by the molecular weight of the sample. Additionally, the introduction of X-ray free-electron lasers (XFELs) for studying the structure and dynamics of biological macromolecules has the potential to overcome the effects of radiation damage (Spence, 2017), providing new opportunities for crystallography and imaging at atomic resolution on timescales from femtoseconds to seconds (serial femtosecond crystallography/SFX) (Chapman et al, 2011;Coe & Fromme, 2016).…”