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1. #AIR CREATIVE COLLECTION INSTALLER PT10 MANUAL#
2. #AIR CREATIVE COLLECTION INSTALLER PT10 SOFTWARE#
3. #AIR CREATIVE COLLECTION INSTALLER PT10 SERIES#

The reaction then proceeds until it is stopped by vitrification of the sample. Mixing can either be achieved within custom-built microfluidic devices (Kaledhonkar et al., 2018 ▸ Mäeots et al., 2020 ▸) or on the grid (Berriman & Unwin, 1994 ▸ Dandey et al., 2020 ▸). The reaction is typically initiated by rapid mixing at a defined time point before vitrification.

#AIR CREATIVE COLLECTION INSTALLER PT10 SOFTWARE#

These include software developments to account for continuous flexibility (Nakane et al., 2018 ▸), the improved signal to noise obtained with modern microscope hardware, and developments in fast cryo-EM grid preparation (Feng et al., 2017 ▸ Rubinstein et al., 2019 ▸ Jain et al., 2012 ▸). More recent advances in biological cryo-EM promise to make this method more broadly applicable. Unwin first demonstrated this approach of rapid mixing, freeze-quenching and EM image processing to study the active state of the acetyl­choline receptor in the mid-1990s (Unwin, 1995 ▸). With fast grid-preparation methods, however, a reaction may be initiated and quenched with a very short and defined time delay (>5 ms). This typically limits the technique to time points longer than ∼10 s.

#AIR CREATIVE COLLECTION INSTALLER PT10 MANUAL#

In principle, conventional grid-making approaches allow time-resolved studies, but typical blotting is inherently slow because of the manual sample application and the relatively long blot times (seconds). Cryo-electron microscopy (cryo-EM) is well suited for such studies: conformations may be separated in silico if they are structurally different and present in sufficient number upon vitrification.

#AIR CREATIVE COLLECTION INSTALLER PT10 SERIES#

Such reactions often involve a series of transient intermediate states which can be trapped and analysed if the system is studied with appropriate temporal resolution. Motor proteins and many other biological macromolecules change conformations, form complexes or dissociate from their interaction partners as part of their functional cycle. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development. 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. 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. 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. 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. 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. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. 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.