Serial femtosecond and serial synchrotron crystallography yield data of equivalent quality: a systematic comparison

Abstract

For the two proteins myoglobin (MB) and fluoroacetate dehalogenase (FAcD), we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of crystals, the same sample delivery device, as well as the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality. For both proteins reasonable data statistics can be obtained with approximately 5000 room temperature diffraction images irrespective of the radiation source. The direct comparability of SSX and SFX data indicates that diffraction quality is rather linked to the properties of the crystals than to the radiation source. Time-resolved experiments can therefore be conducted at the source that best matches the desired time-resolution.

Best practices for time-resolved serial synchrotron crystallography

“…In spite of many recent improvements, serial crystallography experiments are still very sample-demanding, and this is even more the case for time-resolved studies where several structures are to be obtained, each of which requires at least $5000 diffraction patterns (Moreno-Chicano et al, 2019;Weinert et al, 2017;Mehrabi et al, 2021;Gorel et al, 2021). Thus, a critical question is whether a sufficient amount of protein is available.…”

“…Arguably the first consideration when assessing the quality of any data set should be completeness; this single parameter is generally applicable to any crystallographic experiment and provides context for all other data-quality parameters. If the completeness of the data set is below an approximate cutoff of 95% then the meaning that can be derived from the scaling statistics is limited (Mehrabi et al, 2021). The pertinent question when collecting data may then appear to be 'how many diffraction images do I need to collect to obtain a complete data set?'.…”

“…The amount of data needed is ultimately dependent on both the sample and the experimental design. Although the minimum reported value for a good-quality data set is $5000 indexable diffraction patterns, this number certainly increases the less populated the state one wants to observe is (Weinert et al, 2017;Moreno-Chicano et al, 2019;Mehrabi et al, 2021). Therefore, in order to determine when an experiment is complete, it is beneficial to monitor the hit rate (i.e.…”

Serial Electron Diffraction Data Processing With diffractem and CrystFEL

“…While rotation crystallography, whether using electrons or X-rays, can yield high-quality crystallographic data from nanometric crystals, an inherent limitation is the accumulation of radiation damage during rotation data collection ( Hattne et al, 2018 ), prohibiting acquisition of damage-minimized data. On the other hand, damage accumulation is evaded in serial crystallography, where each crystal is exposed once, using femtosecond X-ray pulses at extreme intensities that record diffraction data before Coulomb explosion ( Chapman et al, 2011 ), or X-ray/electron pulses at lower intensity below a critical dose threshold, which can yield equivalent results ( Bücker et al, 2020 ; Mehrabi et al, 2020 ).…”

Serial Electron Crystallography: New Developments for Data Collection and Analysis

“…Serial electron diffraction (SerialED) (Smeets, Zou and Wan, 2018;Bücker et al, 2020) is an emerging threedimensional electron diffraction (3D-ED) method (Gemmi et al, 2019;Nannenga and Gonen, 2019), where data from a large ensemble of macromolecular or small-molecule nano-crystals is merged into a high-resolution structure solution. Like in serial X-ray crystallography at synchrotrons and X-ray free-electron lasers (Mehrabi et al, 2020), SerialED is typically performed in the limit of rapidly acquired single or dose-fractionated diffraction shots per diffracting volume, minimizing deleterious effects of radiation damage. However, the flexibility of a scanning TEM (S/TEM) enables a much broader range of data collection strategies, blurring the lines between serial crystallography (SX), scanning electron nano-diffraction (SEND), and massively automated rotation electron diffraction (RED).…”