ArticleActa crystallographica. Section D, Structural biology2021
On-grid and in-flow mixing for time-resolved cryo-EM.
Article in Acta crystallographica. Section D, Structural biology, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed.
- Article
- EasyGrid: a versatile platform for automated cryo-EM sample preparation and quality control.Nature methods · 2026Article
- Tool antibody fragments reveal multiple conformations of the rhodopsin-Gi signaling complex.Biophysical journal · 2026Article
- Microfluidic Devices for High-Throughput In Situ Serial Crystallography.Advances in biochemical engineering/biotechnology · 2026Review
- Microsecond Time-Resolved Cryo-EM Based on Jet Vitrification.bioRxiv : the preprint server for biology · 2025Article
- Article
- Advancing time-resolved structural biology: latest strategies in cryo-EM and X-ray crystallography.Nature methods · 2025Review
- FLUID-CELL: Flow-enabled Light and Ultrastructural Imaging Device for Correlative Electron and Light Localization.bioRxiv : the preprint server for biology · 2025Article
- Advances in microfluidic mixers for time-resolved structural biology with X-rays.Biophysical reviews · 2025Review
- Article
- A PDMS-based Microfluidic Chip Assembly for Time-Resolved Cryo-EM (TRCEM) Sample Preparation.Bio-protocol · 2025Article
- Time-resolved cryo-EM (TRCEM) sample preparation using a PDMS-based microfluidic chip assembly.bioRxiv : the preprint server for biology · 2024Article
- Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription.Nature communications · 2024Article
- Article
- Article
- Time resolution in cryo-EM using a novel PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.bioRxiv : the preprint server for biology · 2023Article
- Frozen in time: analyzing molecular dynamics with time-resolved cryo-EM.Structure (London, England : 1993) · 2023Review
- Evaluation of correlated studies using liquid cell and cryo-transmission electron microscopy: Hydration of calcium sulphate and the phase transformation pathways of bassanite to gypsum.Journal of microscopy · 2022Article
- Towards sub-millisecond cryo-EM grid preparation.Faraday discussions · 2022Article
- Structural dynamics: review of time-resolved cryo-EM.Acta crystallographica. Section D, Structural biology · 2022Review
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4 authors.
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Abstract
Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5-10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7-700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air-water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.
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