Rapid melting and revitrification as an approach to microsecond time-resolved cryo-electron microscopy

Voss, Jonathan M.; Harder, Oliver F.; Olshin, Pavel K.; Drabbels, Marcel; Lorenz, Ulrich J.

doi:10.1016/j.cplett.2021.138812

Cited by 33 publications

(47 citation statements)

References 44 publications

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“…Heat transfer simulations confirm that its size closely matches that of the area whose temperature exceeds the melting point by the end of the laser pulse, as discussed in more detail below. The crystallization of the surrounding areas likely does not occur while the sample cools, since the cooling rate of >10 7 K/s is much higher than is needed to outrun crystallization, as previously shown, 18 which allows the sample to revitrify in the center of the laser focus. Instead, it appears that crystallization already occurs during laser heating, as the sample traverses “no man's land”.…”

Section: Resultsmentioning

confidence: 95%

“…2(g) and 2(n) ], allowing us to identify the power at which the sample reaches the melting point of ice. 18 To heat the sample to a specific temperature exceeding the melting point, the laser power can be adjusted by comparison with heat transfer simulations, as discussed below.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently demonstrated a novel approach to time-resolved cryo-EM with microsecond time resolution, three orders of magnitude faster than previously possible. 18,19 Our method involves melting a cryo sample with a laser beam for a duration of tens of microseconds or longer, which provides a tunable time window during which a stimulus can cause the particles to undergo conformational dynamics. When the heating laser is switched off, the sample revitrifies within just a few microseconds, 18 trapping the particles in their transient configurations, which can be subsequently imaged with the electron beam.…”

Section: Introductionmentioning

confidence: 99%

“… 18,19 Our method involves melting a cryo sample with a laser beam for a duration of tens of microseconds or longer, which provides a tunable time window during which a stimulus can cause the particles to undergo conformational dynamics. When the heating laser is switched off, the sample revitrifies within just a few microseconds, 18 trapping the particles in their transient configurations, which can be subsequently imaged with the electron beam. A short laser pulse can serve as a suitable stimulus to induce dynamics, either by directly triggering a photoactive protein or by releasing a caged compound, such as ions, ATP, or peptides.…”

Section: Introductionmentioning

confidence: 99%

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Microsecond melting and revitrification of cryo samples

Voss

Harder

Olshin

et al. 2021

Structural Dynamics

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The dynamics of proteins that are associated with their function typically occur on the microsecond timescale, orders of magnitude faster than the time resolution of cryo-electron microscopy. We have recently introduced a novel approach to time-resolved cryo-electron microscopy that affords microsecond time resolution. It involves melting a cryo sample with a heating laser, so as to allow dynamics of the proteins to briefly occur in the liquid phase. When the laser is turned off, the sample rapidly revitrifies, trapping the particles in their transient configurations. Precise control of the temperature evolution of the sample is crucial for such an approach to succeed. Here, we provide a detailed characterization of the heat transfer occurring under laser irradiation as well as the associated phase behavior of the cryo sample. While areas close to the laser focus undergo melting and revitrification, surrounding regions crystallize. In situ observations of these phase changes therefore provide a convenient approach for assessing the temperature reached in each melting and revitrification experiment and for adjusting the heating laser power on the fly.

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Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Microsecond melting and revitrification of cryo samples

Voss

Harder

Olshin

et al. 2021

Structural Dynamics

Self Cite

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show abstract

“…Recently, new methods in cryoEM demonstrate the potential for time-resolved measurements and '4D reconstructions'. Progress toward this goal has been made in sample preparation, microscope hardware, data collection, and analysis techniques [79,80,[89][90][91][92][93][94][95][96][97]].…”

Section: Time-resolved Cryoemmentioning

confidence: 99%

Challenges and approaches to studying pore-forming proteins

Benton

Bayly-Jones

2021

Biochemical Society Transactions

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Pore-forming proteins (PFPs) are a broad class of molecules that comprise various families, structural folds, and assembly pathways. In nature, PFPs are most often deployed by their host organisms to defend against other organisms. In humans, this is apparent in the immune system, where several immune effectors possess pore-forming activity. Furthermore, applications of PFPs are found in next-generation low-cost DNA sequencing, agricultural crop protection, pest control, and biosensing. The advent of cryoEM has propelled the field forward. Nevertheless, significant challenges and knowledge-gaps remain. Overcoming these challenges is particularly important for the development of custom, purpose-engineered PFPs with novel or desired properties. Emerging single-molecule techniques and methods are helping to address these unanswered questions. Here we review the current challenges, problems, and approaches to studying PFPs.

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Other Structural Biology Techniques

Sachleben

2023

Dynamics and Kinetics in Structural Biology

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Rapid melting and revitrification as an approach to microsecond time-resolved cryo-electron microscopy

Cited by 33 publications

References 44 publications

Microsecond melting and revitrification of cryo samples

Microsecond melting and revitrification of cryo samples

Challenges and approaches to studying pore-forming proteins

Other Structural Biology Techniques

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