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

Abstract

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.

Hybrid Plasma/Molecular-Dynamics Approach for Efficient XFEL Radiation Damage Simulations

“…We show that the gain in computational speed provided by plasma/MD model can be leveraged to estimate the radiation damage induced disulfide (S-S) bond elongation on the example of a lysozyme nano-crystal. This effect has recently been demonstrated in an X-ray pump/X-ray probe serial femtosecond crystallography (SFX) experiment [23]. It was shown in that work that the observed bond length depends on the fluence of the incident pump pulse as well as the time delay between the pump and probe pulses.…”

“…The electron gas temperature can then be used to solve for the temperature of the ions as a function of time [26]. It is assumed that electrons added to the gas are instantly thermalized, which may lead to an overestimation of the secondary ionization caused by the gas [23].…”

High-throughput in situ experimental phasing

“…However, very high quality diffraction data from significantly smaller crystals, including submicrometresized samples, are often achievable using SFX methods (Gati et al, 2017;Nass, Redecke et al, 2020). Consequently, various sample-presentation strategies have been developed to minimize background noise for SFX, because nanometre-sized to micrometre-sized crystals produce weaker diffraction data compared with traditionally sized samples (Awel et al, 2018;Beyerlein et al, 2017;Calvey et al, 2019;Dasgupta et al, 2019;Davy et al, 2019;Doak et al, 2018;Echelmeier et al, 2019;Fuller et al, 2017;Grunbein & Nass Kovacs, 2019;Lieske et al, 2019;Martiel et al, 2019;Meents et al, 2017;Mehrabi et al, 2019;Monteiro et al, 2019;Nass, Gorel et al, 2020;Nogly et al, 2016;Oberthuer et al, 2017;Orville, 2017;Owen et al, 2017;Roedig et al, 2017;Schulz et al, 2018Schulz et al, , 2019Shelby et al, 2020;Sierra et al, 2016;Stagno et al, 2017;Suga et al, 2020;Sugahara et al, 2017;Weinert et al, 2017;Wiedorn et al, 2018;Zhao et al, 2019). In addition, X-ray pulse durations of tens of femtoseconds and the tight focus and intensity of XFEL pulses often produce very high-quality data sets with little or no radiation-induced effects in the data and refined atomic models.…”

Determination of the Structure of Biological Macromolecular Particles Using X-Ray Lasers. Achievements and Prospects

“…Локальные разрушения наблюдались при длине импульса 40 фс [72]. Наблюдались изменения электронной плотности для атомов или ионов металлов, атомов серы, структурные изменения в области, окружающей кластер с тяжелыми атомами [72][73][74], удлинение дисульфидных связей и структурные изменения боковых групп ароматических остатков [75].…”

“…Во многих недавних исследованиях в этой области применяется используемая на современных лазерах схема pump-probe эксперимента, при которой на образец подается два лазерных импульса через фиксированный интервал времени. Первый импульс инициирует процессы разрушения в образце, а второй импульс предназначен для получения дифракционной картины для образца, в котором эти разрушения уже начали происходить между двумя импульсами [75,76]. Заметим, что в недавнем исследовании локальные эффекты радиационного разрушения в кристаллах наблюдались уже при интервалах времени между импульсами, приблизительно равными 20-30 фс [75].…”