Influence of pump laser fluence on ultrafast myoglobin structural dynamics.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

Dynamic catcher for stabilization of high-viscosity extrusion jets.

A 'catcher' based on a revolving cylindrical collector is described. The simple and inexpensive device reduces free-jet instabilities inherent to high-viscosity extrusion injection, facilitating delivery of microcrystals for serial diffraction X-ray crystallography.

Structures of an unusual 3-hydroxyacyl dehydratase (FabZ) from a ladderane-producing organism with an unexpected substrate preference.

The genomes of anaerobic ammonium-oxidizing (anammox) bacteria contain a gene cluster comprising genes of unusual fatty acid biosynthesis enzymes that were suggested to be involved in the synthesis of the unique "ladderane" lipids produced by these organisms. This cluster encodes an acyl carrier protein (denoted as "amxACP") and a variant of FabZ, an ACP-3-hydroxyacyl dehydratase. In this study, we characterize this enzyme, which we call anammox-specific FabZ ("amxFabZ"), to investigate the unresolved biosynthetic pathway of ladderane lipids. We find that amxFabZ displays distinct sequence differences to "canonical" FabZ, such as a bulky, apolar residue on the inside of the substrate-binding tunnel, where the canonical enzyme has a glycine. Additionally, substrate screens suggest that amxFabZ efficiently converts substrates with acyl chain lengths of up to eight carbons, whereas longer substrates are converted much more slowly under the conditions used. We also present crystal structures of amxFabZs, mutational studies and the structure of a complex between amxFabZ and amxACP, which show that the structures alone cannot explain the apparent differences from canonical FabZ. Moreover, we find that while amxFabZ does dehydrate substrates bound to amxACP, it does not convert substrates bound to canonical ACP of the same anammox organism. We discuss the possible functional relevance of these observations in the light of proposals for the mechanism for ladderane biosynthesis.

Growing and making nano- and microcrystals.

Thanks to recent technological advances in X-ray and micro-electron diffraction and solid-state NMR, structural information can be obtained by using much smaller crystals. Thus, microcrystals have become a valuable commodity rather than a mere stepping stone toward obtaining macroscopic crystals. Microcrystals are particularly useful for structure determination using serial data collection approaches at synchrotrons and X-ray free-electron lasers. The latter's enormous peak brilliance and short X-ray pulse duration mean that structural information can be obtained before the effects of radiation damage are seen; these properties also facilitate time-resolved crystallography. To establish defined reaction initiation conditions, microcrystals with a desired and narrow size distribution are critical. Here, we describe milling and seeding techniques as well as filtration approaches for the reproducible and size-adjustable preparation of homogeneous nano- and microcrystals. Nanocrystals and crystal seeds can be obtained by milling using zirconium beads and the BeadBug homogenizer; fragmentation of large crystals yields micro- or nanocrystals by flowing crystals through stainless steel filters by using an HPLC pump. The approaches can be scaled to generate micro- to milliliter quantities of microcrystals, starting from macroscopic crystals. The procedure typically takes 3-5 d, including the time required to grow the microcrystals.

Is orange carotenoid protein photoactivation a single-photon process?

In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCPR, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCPR formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCPR.

Time-resolved serial femtosecond crystallography on fatty-acid photodecarboxylase: lessons learned.

Upon absorption of a blue-light photon, fatty-acid photodecarboxylase catalyzes the decarboxylation of free fatty acids to form hydrocarbons (for example alkanes or alkenes). The major components of the catalytic mechanism have recently been elucidated by combining static and time-resolved serial femtosecond crystallography (TR-SFX), time-resolved vibrational and electronic spectroscopies, quantum-chemical calculations and site-directed mutagenesis [Sorigué et al. (2021), Science, 372, eabd5687]. The TR-SFX experiments, which were carried out at four different picosecond to microsecond pump-probe delays, yielded input for the calculation of Fourier difference maps that demonstrated light-induced decarboxylation. Here, some of the difficulties encountered during the experiment as well as during data processing are highlighted, in particular regarding space-group assignment, a pump-laser power titration is described and data analysis is extended by structure-factor extrapolation of the TR-SFX data. Structure refinement against extrapolated structure factors reveals a reorientation of the generated hydrocarbon and the formation of a photoproduct close to Cys432 and Arg451. Identification of its chemical nature, CO2 or bicarbonate, was not possible because of the limited data quality, which was assigned to specificities of the crystalline system. Further TR-SFX experiments on a different crystal form are required to identify the photoproducts and their movements during the catalytic cycle.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Crystallographic Studies of Rhodopsins: Structure and Dynamics.

Crystal structures have provided detailed insight in the architecture of rhodopsin photoreceptors. Of particular interest are the protein-chromophore interactions that govern the light-induced retinal isomerization and ultimately induce the large structural changes important for the various biological functions of the family. The reaction intermediates occurring along the rhodopsin photocycle have vastly differing lifetimes, from hundreds of femtoseconds to milliseconds. Detailed insight at high spatial and temporal resolution can be obtained by time-resolved crystallography using pump-probe approaches at X-ray free-electron lasers. Alternatively, cryotrapping approaches can be used. Both the approaches are described, including illumination and sample delivery. The importance of appropriate photoexcitation avoiding multiphoton absorption is stressed.

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: Impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP.

Unifying Perspective of the Ultrafast Photodynamics of Orange Carotenoid Proteins from Synechocystis: Peril of High-Power Excitation, Existence of Different S* States, and Influence of Tagging.

A substantial number of Orange Carotenoid Protein (OCP) studies have aimed to describe the evolution of singlet excited states leading to the formation of a photoactivated form, OCPR. The most recent one suggests that 3 ps-lived excited states are formed after the sub-100 fs decay of the initial S2 state. The S* state, which has the longest reported lifetime of a few to tens of picoseconds, is considered to be the precursor of the first red photoproduct P1. Here, we report the ultrafast photodynamics of the OCP from Synechocystis PCC 6803 carried out using visible-near infrared femtosecond time-resolved absorption spectroscopy as a function of the excitation pulse power and wavelength. We found that a carotenoid radical cation can form even at relatively low excitation power, obscuring the determination of photoactivation yields for P1. Moreover, the comparison of green (540 nm) and blue (470 nm) excitations revealed the existence of an hitherto uncharacterized excited state, denoted as S∼, living a few tens of picoseconds and formed only upon 470 nm excitation. Because neither the P1 quantum yield nor the photoactivation speed over hundreds of seconds vary under green and blue continuous irradiation, this S∼ species is unlikely to be involved in the photoactivation mechanism leading to OCPR. We also addressed the effect of His-tagging at the N- or C-termini on the excited-state photophysical properties. Differences in spectral signatures and lifetimes of the different excited states were observed at a variance with the usual assumption that His-tagging hardly influences protein dynamics and function. Altogether our results advocate for the careful consideration of the excitation power and His-tag position when comparing the photoactivation of different OCP variants and beg to revisit the notion that S* is the precursor of photoactivated OCPR.

Serial femtosecond crystallography.

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 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 beamtime 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.

Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex.

Nitrate is an abundant nutrient and electron acceptor throughout Earth's biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein's electron transport chain, and elucidate the electron transfer pathways within the complex.

Discerning best practices in XFEL-based biological crystallography - standards for nonstandard experiments.

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a novel tool in structural biology. In contrast to conventional crystallography, SFX relies on merging partial intensities acquired with X-ray beams of often randomly fluctuating properties from a very large number of still diffraction images of generally randomly oriented microcrystals. For this reason, and possibly due to limitations of the still evolving data-analysis programs, XFEL-derived SFX data are typically of a lower quality than 'standard' crystallographic data. In contrast with this, the studies performed at XFELs often aim to investigate issues that require precise high-resolution data, for example to determine structures of intermediates at low occupancy, which often display very small conformational changes. This is a potentially dangerous combination and underscores the need for a critical evaluation of procedures including data-quality standards in XFEL-based structural biology. Here, such concerns are addressed.

Effect of X-ray free-electron laser-induced shockwaves on haemoglobin microcrystals delivered in a liquid jet.

X-ray free-electron lasers (XFELs) enable obtaining novel insights in structural biology. The recently available MHz repetition rate XFELs allow full data sets to be collected in shorter time and can also decrease sample consumption. However, the microsecond spacing of MHz XFEL pulses raises new challenges, including possible sample damage induced by shock waves that are launched by preceding pulses in the sample-carrying jet. We explored this matter with an X-ray-pump/X-ray-probe experiment employing haemoglobin microcrystals transported via a liquid jet into the XFEL beam. Diffraction data were collected using a shock-wave-free single-pulse scheme as well as the dual-pulse pump-probe scheme. The latter, relative to the former, reveals significant degradation of crystal hit rate, diffraction resolution and data quality. Crystal structures extracted from the two data sets also differ. Since our pump-probe attributes were chosen to emulate EuXFEL operation at its 4.5 MHz maximum pulse rate, this prompts concern about such data collection.

Illumination guidelines for ultrafast pump-probe experiments by serial femtosecond crystallography.

Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.

Structural dynamics in proteins induced by and probed with X-ray free-electron laser pulses.

X-ray free-electron lasers (XFELs) enable crystallographic structure determination beyond the limitations imposed upon synchrotron measurements by radiation damage. The need for very short XFEL pulses is relieved through gating of Bragg diffraction by loss of crystalline order as damage progresses, but not if ionization events are spatially non-uniform due to underlying elemental distributions, as in biological samples. Indeed, correlated movements of iron and sulfur ions were observed in XFEL-irradiated ferredoxin microcrystals using unusually long pulses of 80 fs. Here, we report a femtosecond time-resolved X-ray pump/X-ray probe experiment on protein nanocrystals. We observe changes in the protein backbone and aromatic residues as well as disulfide bridges. Simulations show that the latter's correlated structural dynamics are much slower than expected for the predicted high atomic charge states due to significant impact of ion caging and plasma electron screening. This indicates that dense-environment effects can strongly affect local radiation damage-induced structural dynamics.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.

Ab initio structure determination from experimental fluctuation X-ray scattering data.

Fluctuation X-ray scattering (FXS) is an emerging experimental technique in which X-ray solution scattering data are collected from particles in solution using ultrashort X-ray exposures generated by a free-electron laser (FEL). FXS experiments overcome the low data-to-parameter ratios associated with traditional solution scattering measurements by providing several orders of magnitude more information in the final processed data. Here we demonstrate the practical feasibility of FEL-based FXS on a biological multiple-particle system and describe data-processing techniques required to extract robust FXS data and significantly reduce the required number of snapshots needed by introducing an iterative noise-filtering technique. We showcase a successful ab initio electron density reconstruction from such an experiment, studying the Paramecium bursaria Chlorella virus (PBCV-1).