Probing the modulation of enzyme kinetics by multi-temperature, time-resolved serial crystallography

Mehrabi, P.; Stetten, David von; Leimkohl, Jan-Philipp; Tellkamp, Friedjof; Schulz, Eike C.

doi:10.1101/2021.11.07.467596

Cited by 8 publications

(7 citation statements)

References 81 publications

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“…tural changes in response to temperature (Mehrabi et al, 2021). These experiments could again benefit from the strategy presented in this study, since the crystals are directly grown in different environments and not confronted with environment changes after growth, reducing stress on the crystal and thus potentially increasing diffraction quality.…”

Section: Discussionmentioning

confidence: 99%

JINXED: just in time crystallization for easy structure determination of biological macromolecules

Henkel¹,

Galchenkova²,

Maracke³

et al. 2023

IUCrJ

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Macromolecular crystallography is a well established method in the field of structural biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now under development towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand-soaking and cryo-protection. These handling steps can cause significant crystal damage, and hence reduce data quality. Furthermore, in time-resolved experiments based on serial crystallography, which use micrometre-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method that combines protein crystallization and data collection in a novel one-step process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg-white lysozyme and crystallization times of only a few seconds. This method, called JINXED (Just IN time Crystallization for Easy structure Determination), promises high-quality data due to the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

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Section: Discussionmentioning

confidence: 99%

JINXED: just in time crystallization for easy structure determination of biological macromolecules

Henkel¹,

Galchenkova²,

Maracke³

et al. 2023

IUCrJ

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show abstract

“…Here, we showed that sample consumption per dataset using this system is en par or even lower than with fixed-target (Roedig et al, 2017;Schulz et al, 2018) or viscous-extrusion approaches (Botha et al, 2018) and benefits directly from optimization of crystallization to achieve a high yield of homogenous microcrystals. At the same time, the TapeDrive system allows for uninterrupted data collection without manual intervention or the need to enter the hutch, enabling future automated multiparameter data collection for multidimensional serial crystallography (Mehrabi et al, 2021). This was demonstrated by collecting 40 datasets in less than 30 min, wherein each dataset had good statistics and usable diffraction to a resolution of better than 2 A ˚and each dataset took 40 s to collect with a PILATUS 6M detector, corresponding to only 7.5 s using an EIGER 16M (Dectris, Switzerland) at 133 Hz.…”

Section: Discussionmentioning

confidence: 99%

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

Zielinski¹,

Andaleeb²,

Bui³

et al. 2022

IUCrJ

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Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

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“…Moreover, the addition of temperature control to the TapeDrive might facilitate rapid crystallization for proteins whose solubility is temperature dependent. On that note, recently multi-dimensional studies became apparent in serial crystallography to investigate structural changes as response to temperature changes (Mehrabi et al, 2021). Here, the experiment could again benefit from the strategy presented in this study, since the crystals are directly grown in the different environments and not confronted with environment changes after growth, reducing stress on the crystal lattice and thus potentially increasing diffraction quality.…”

Section: Discussionmentioning

confidence: 99%

JINXED: Just in time crystallization for easy structure determination of biological macromolecules

Henkel

Galchenkova

Maracke

et al. 2022

Preprint

View full text Add to dashboard Cite

Macromolecular crystallography is a well-established method in the field of structure biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now developing towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand soaking and cryo-protection. These handling steps can cause significant crystal damage, causing a decrease in data quality. Furthermore, in time-resolved experiments based on serial crystallography that use micron-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method combining protein crystallization and data collection in a novel one-step-process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg white lysozyme and crystallization times of only a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

show abstract

Probing the modulation of enzyme kinetics by multi-temperature, time-resolved serial crystallography

Cited by 8 publications

References 81 publications

JINXED: just in time crystallization for easy structure determination of biological macromolecules

JINXED: just in time crystallization for easy structure determination of biological macromolecules

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

JINXED: Just in time crystallization for easy structure determination of biological macromolecules

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