Low-pass spectral analysis of time-resolved serial femtosecond crystallography data.

Low-pass spectral analysis (LPSA) is a recently developed dynamics retrieval algorithm showing excellent retrieval properties when applied to model data affected by extreme incompleteness and stochastic weighting. In this work, we apply LPSA to an experimental time-resolved serial femtosecond crystallography (TR-SFX) dataset from the membrane protein bacteriorhodopsin (bR) and analyze its parametric sensitivity. While most dynamical modes are contaminated by nonphysical high-frequency features, we identify two dominant modes, which are little affected by spurious frequencies. The dynamics retrieved using these modes shows an isomerization signal compatible with previous findings. We employ synthetic data with increasing timing uncertainty, increasing incompleteness level, pixel-dependent incompleteness, and photon counting errors to investigate the root cause of the high-frequency contamination of our TR-SFX modes. By testing a range of methods, we show that timing errors comparable to the dynamical periods to be retrieved produce a smearing of dynamical features, hampering dynamics retrieval, but with no introduction of spurious components in the solution, when convergence criteria are met. Using model data, we are able to attribute the high-frequency contamination of low-order dynamical modes to the high levels of noise present in the data. Finally, we propose a method to handle missing observations that produces a substantial dynamics retrieval improvement from synthetic data with a significant static component. Reprocessing of the bR TR-SFX data using the improved method yields dynamical movies with strong isomerization signals compatible with previous findings.

Correction of rhodopsin serial crystallography diffraction intensities for a lattice-translocation defect.

Rhodopsin is a G-protein-coupled receptor that detects light and initiates the intracellular signalling cascades that underpin vertebrate vision. Light sensitivity is achieved by covalent linkage to 11-cis retinal, which isomerizes upon photo-absorption. Serial femtosecond crystallography data collected from rhodopsin microcrystals grown in the lipidic cubic phase were used to solve the room-temperature structure of the receptor. Although the diffraction data showed high completeness and good consistency to 1.8 Šresolution, prominent electron-density features remained unaccounted for throughout the unit cell after model building and refinement. A deeper analysis of the diffraction intensities uncovered the presence of a lattice-translocation defect (LTD) within the crystals. The procedure followed to correct the diffraction intensities for this pathology enabled the building of an improved resting-state model. The correction was essential to both confidently model the structure of the unilluminated state and interpret the light-activated data collected after photo-excitation of the crystals. It is expected that similar cases of LTD will be observed in other serial crystallography experiments and that correction will be required in a variety of systems.

Ultrafast structural changes direct the first molecular events of vision.

Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

Dynamics retrieval from stochastically weighted incomplete data by low-pass spectral analysis.

Time-resolved serial femtosecond crystallography (TR-SFX) provides access to protein dynamics on sub-picosecond timescales, and with atomic resolution. Due to the nature of the experiment, these datasets are often highly incomplete and the measured diffracted intensities are affected by partiality. To tackle these issues, one established procedure is that of splitting the data into time bins, and averaging the multiple measurements of equivalent reflections within each bin. This binning and averaging often involve a loss of information. Here, we propose an alternative approach, which we call low-pass spectral analysis (LPSA). In this method, the data are projected onto the subspace defined by a set of trigonometric functions, with frequencies up to a certain cutoff. This approach attenuates undesirable high-frequency features and facilitates retrieving the underlying dynamics. A time-lagged embedding step can be included prior to subspace projection to improve the stability of the results with respect to the parameters involved. Subsequent modal decomposition allows to produce a low-rank description of the system's evolution. Using a synthetic time-evolving model with incomplete and partial observations, we analyze the LPSA results in terms of quality of the retrieved signal, as a function of the parameters involved. We compare the performance of LPSA to that of a range of other sophisticated data analysis techniques. We show that LPSA allows to achieve excellent dynamics reconstruction at modest computational cost. Finally, we demonstrate the superiority of dynamics retrieval by LPSA compared to time binning and merging, which is, to date, the most commonly used method to extract dynamical information from TR-SFX data.

Structure-factor amplitude reconstruction from serial femtosecond crystallography of two-dimensional membrane-protein crystals.

Serial femtosecond crystallography of two-dimensional membrane-protein crystals at X-ray free-electron lasers has the potential to address the dynamics of functionally relevant large-scale motions, which can be sterically hindered in three-dimensional crystals and suppressed in cryocooled samples. In previous work, diffraction data limited to a two-dimensional reciprocal-space slice were evaluated and it was demonstrated that the low intensity of the diffraction signal can be overcome by collecting highly redundant data, thus enhancing the achievable resolution. Here, the application of a newly developed method to analyze diffraction data covering three reciprocal-space dimensions, extracting the reciprocal-space map of the structure-factor amplitudes, is presented. Despite the low resolution and completeness of the data set, it is shown by molecular replacement that the reconstructed amplitudes carry meaningful structural information. Therefore, it appears that these intrinsic limitations in resolution and completeness from two-dimensional crystal diffraction may be overcome by collecting highly redundant data along the three reciprocal-space axes, thus allowing the measurement of large-scale dynamics in pump-probe experiments.

Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals.

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump-probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.

Direct visualization of a Fe(IV)-OH intermediate in a heme enzyme.

Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)-OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)-OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)-OH in a peroxidase.

Heme enzymes. Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase.

Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O-O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.