Science with X-ray Free-Electron Lasers (XFELs)
Many biological processes of functional relevance, enzyme catalysis, redox reactions, ligand binding, and allosteric regulation, are intrinsically dynamic and occur far from equilibrium. Conventional structural biology approaches, largely based on cryogenic and static measurements, often fail to capture these transient and heterogeneous states.
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X-ray Free-Electron Lasers (XFELs) enable structural studies of biomolecules under ambient conditions and on ultrafast time scales, providing direct access to reaction intermediates, conformational heterogeneity, and functional asymmetry. This capability allows us to move beyond static structures and towards a mechanistic understanding of biomolecular function.
Why XFELs?
XFELs produce extremely intense, femtosecond X-ray pulses that allow diffraction data to be collected before the onset of radiation damage (“diffraction-before-destruction”). This enables high-quality structural measurements from micro- and nanocrystals at room temperature.
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Importantly, XFELs allow time-resolved experiments in which biochemical reactions are initiated and probed at defined delay times, providing structural snapshots along reaction pathways. As a result, XFELs uniquely enable the direct observation of transient states that are inaccessible to equilibrium or cryogenic approaches.
Experimental approaches
Serial Femtosecond Crystallography (SFX)
SFX enables structure determination from microcrystals delivered serially across an XFEL beam. Each crystal is exposed to a single femtosecond X-ray pulse, allowing diffraction data to be collected before the crystals are destroyed. This approach permits measurements at room temperature and from crystals that are too small or radiation-sensitive for conventional crystallography. By combining large numbers of single-shot diffraction patterns, SFX provides high-quality structural information while preserving conformational heterogeneity and dynamic features that are often suppressed under cryogenic conditions.
Time-resolved SFX (TR-SFX)
In TR-SFX, reactions are initiated directly in crystallo and probed by femtosecond XFEL pulses at defined delay times (Δt). Structural snapshots collected at successive time points capture transient intermediates and conformational changes, enabling reconstruction of enzymatic reaction pathways. ​Reaction initiation can be achieved through different triggering strategies, including rapid mixing of substrates or ligands (mix-and-inject), optical excitation in pump–probe experiments, or other perturbations. The chosen triggering method defines the accessible time window, spanning from femtoseconds to seconds.
From static structures to functional ensembles
XFEL-based structural biology shifts the focus from single, static structures to ensembles of conformations sampled under functional conditions. This is particularly relevant for multimeric enzymes, redox-active proteins, and systems displaying cooperativity or functional asymmetry.
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Room-temperature and time-resolved XFEL measurements reveal how conformational heterogeneity, flexibility, and transient interactions contribute to biological function, enabling mechanistic insights that cannot be inferred from static or cryogenic structures alone.
Integrative structural biology
XFEL experiments are most powerful when integrated with complementary approaches, including solution biophysics, spectroscopy, kinetics, and molecular simulations. Structural snapshots obtained at XFELs provide a framework for interpreting functional measurements and for testing mechanistic hypotheses across multiple time and length scales.
International XFEL context
Experiments are conducted at leading international X-ray free-electron laser (XFEL) facilities, providing access to state-of-the-art instrumentation for serial and time-resolved structural biology. These large-scale research infrastructures enable experiments that combine ultrafast time resolution, high X-ray brilliance, and advanced sample-delivery and detection systems, which are essential for studying radiation-sensitive biological samples and non-equilibrium processes. Working within this international XFEL ecosystem allows the integration of complementary expertise in instrumentation, data acquisition, and analysis, and supports the development and application of advanced methodologies for probing biomolecular dynamics under functional conditions.
Scientific perspective
XFELs enable a fundamentally different approach to structural biology, one that treats dynamics, heterogeneity, and non-equilibrium behavior as central features of biological function. By exploiting these capabilities, we aim to uncover mechanistic principles that remain hidden in static structural descriptions.