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

Mehrabi, P.; Bücker, Robert; Bourenkov, Gleb; Ginn, H.; Stetten, David von; Müller‐Werkmeister, Henrike M.; Morizumi, Takefumi; Eger, B.T.; Ou, Wei-Lin; Oghbaey, Saeed; Sarracini, Antoine; Besaw, Jessica E.; ́-Labrosse, O. Pare; Meier, S.; Schikora, H.; Tellkamp, Friedjof; Sherrell, D.A.; Axford, Danny; Owen, R.L.; Ernst, Oliver P.; Pai, E.F.; Schulz, E.C.; Miller, R. J. Dwayne

doi:10.1126/sciadv.abf1380

Cited by 30 publications

(9 citation statements)

References 65 publications

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“…Serial crystallography (SX) is a technique that enables the observation of room temperature structures of various molecules, ranging from macromolecules to small molecules [1][2][3][4] . Unlike conventional X-ray crystallography techniques, SX uses intense X-ray pulses or short X-ray exposure, thus reducing radiation damage to crystal samples [5][6][7] . Moreover, time-resolved chemical and biological reactions of target samples can be observed using pump-probes with photoactivation or liquid composed of a substrate or inhibitor [8][9][10][11][12][13] .…”

Section: Beef Tallow Injection Matrix For Serial Crystallography Ki H...mentioning

confidence: 99%

Beef tallow injection matrix for serial crystallography

Nam

2022

Sci Rep

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Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

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Section: Beef Tallow Injection Matrix For Serial Crystallography Ki H...mentioning

confidence: 99%

Beef tallow injection matrix for serial crystallography

Nam

2022

Sci Rep

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“…It is likely that DTT is rapidly lost due to the vulnerability of the disulfide bond to radiation damage. SSX takes single snapshots of many crystals with limited X-ray exposure per crystal, hence there is much-reduced dose and radiation damage 27 . In our structures, the fresh HGD m crystals incurred an average diffraction-weighted dose (DWD) 28 of 0.19 MGy after 20 ms X-ray exposure 29 .…”

Section: Resultsmentioning

confidence: 99%

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin

Hill,

Nyathi,

Horrell

et al. 2024

Commun Chem

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Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

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“…It is likely that DTT is rapidly lost due to the vulnerability of the disulfide bond to radiation damage. SSX takes single snapshots of many crystals with limited X-ray exposure per crystal, hence there is much reduced dose and radiation damage [22]. In our structures, the fresh HGD crystals incur an average diffraction-weighted dose (DWD) [23] of 0.19 MGy after 20 ms X-ray exposure [24].…”

Section: Resultsmentioning

confidence: 99%

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin

Hill,

Nyathi,

Horrell

et al. 2023

Preprint

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1AbstractHuman gamma-D crystallin (HGD) is the major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin shows no post-translational modifications. After aging for nine months in the absence of light, a covalently bound reducing agent modifying surface cysteines is observed for the first time by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

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

Cited by 30 publications

References 65 publications

Beef tallow injection matrix for serial crystallography

Beef tallow injection matrix for serial crystallography

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin

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