Towards the spatial resolution of metalloprotein charge states by detailed modeling of XFEL crystallographic diffraction

Abstract

Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X-ray absorption edges, which shift to higher energy for progressively more positive valence numbers. Indeed, X-ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X-ray sources at synchrotron facilities with energy resolutions in the range 2-3 Â 10 À4 (ÁE/E). This paper tests whether X-ray free-electron laser (XFEL) pulses, which have a broader bandpass (ÁE/E = 3 Â 10 À3 ) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength-dependent anomalous scattering factors contributed by two differently charged iron centers in the 110-amino-acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X-ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.

Beyond integration: modeling every pixel to obtain better structure factors from stills

“…The maximum-likelihood program presented here, diffBragg, provides a direct protocol for decoupling the various contributions to the scattering which would otherwise obscure the structure factor amplitudes. These noisy contributions include variable per-shot incident photon spectra, crystal morphology and Ewald offset (partiality), all of which can be detrimental in situations involving weak signals, such as anomalous difference amplitudes used for the spatial resolution of heavy atoms (Sauter et al, 2020), or for experimental phasing (Schlichting, 2017). With 2023 shots we achieved similar quality anomalous differences (revealed by CC Ã ano ) to those obtained by the conventional processing of 19 953 shots.…”

“…Pixel-level refinement can resolve extremely sensitive details, such as the dispersion corrections arising from two differently oxidized metal atoms, as shown with synthetic data in the work by Sauter et al (2020). Having potential access to this level of detail from Bragg scattering opens up new and exciting experimental avenues to consider as the next decade of SFX science begins.…”

“…Ultimately, our interest lies in single-electron transfers at the four Mn positions of the PSII catalytic cofactor. Spatially resolving the K absorption edge individually for each Mn center has the potential to elucidate the electronic environment of each Mn atom (Sauter et al, 2020). Such challenging measurements would require quantifying structure factor intensities between Friedel pairs ðjF h j 2 versus jF" h h j 2 ) and among different states at the 1% level of uncertainty.…”

Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

“…UV–vis absorption spectra of a B-type dye decolorizing peroxidase (electron density shown centre) [ 9 ]. Oxidation states of iron sites in a ferredoxin were revealed using diffraction spectroscopy as shown through the use of simulated diffraction patterns [ ∗∗36 ]. Iron Kα XES of methane monooxygenase (MMOH) crystals confirming iron oxidation state [ ∗33 ].…”

Pixel modelling – a new age in SFX data analysis

“…This indicates that diffBragg could have a remarkable impact on future SFX experiments by addressing one of the main bottlenecks of SFX -the need for high amounts of data, translating into large sample quantities. Further, the high accuracy achieved with pixel-level refinement can provide a clear view on extremely sensitive details such as two differently oxidized metal atoms (Sauter et al, 2020). The ability to observe such level of detail expands opportunities for new and exciting experiments to consider for the upcoming years of SFX science.…”