2021
DOI: 10.1107/s2052252521007053
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Millisecond mix-and-quench crystallography (MMQX) enables time-resolved studies of PEPCK with remote data collection

Abstract: Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and the complex experimental infrastructure that are required remain serious obstacles to its widespread application. Here, millisecond mix-and-quench crystallography (MMQX) has been developed, which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, … Show more

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Cited by 9 publications
(6 citation statements)
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“…By contrast, the commercial plunger ‘ Nanuq’ (Mitegen, USA) is specifically designed to vitrify protein crystals but can— to the best of our knowledge —not initiate reactions. More recently a similar crystal cryo-trapping solution was reported 10 . In contrast to the spitrobot , this ‘mix-and-quench’ device relies on non-standard crystal sample holders without bases and thus requires more complex crystal handling under cryo-conditions, which bears the potential for errors.…”
Section: Discussionmentioning
confidence: 85%
See 1 more Smart Citation
“…By contrast, the commercial plunger ‘ Nanuq’ (Mitegen, USA) is specifically designed to vitrify protein crystals but can— to the best of our knowledge —not initiate reactions. More recently a similar crystal cryo-trapping solution was reported 10 . In contrast to the spitrobot , this ‘mix-and-quench’ device relies on non-standard crystal sample holders without bases and thus requires more complex crystal handling under cryo-conditions, which bears the potential for errors.…”
Section: Discussionmentioning
confidence: 85%
“…The plunging velocity is regulated via the applied gas pressure. For typical use we relied on pressure levels from 3 to 6 bar, which enable piston motions on the order of 1.6 m s −1 , comparable to previously published solutions 9 , 10 . The piston is equipped with an electromagnetic SPINE-style sample holder, onto which the micro-meshes are manually mounted.…”
Section: Resultsmentioning
confidence: 99%
“…Also, diffraction resolution is often limited at room temperature. Cryo-trapping methods potentially imply a number of drawbacks: flashcooling and cryoprotection artefacts (for trigger-freeze strategies), altered conformational landscape (for freeze-trigger strategies), specific radiation damage (for synchrotron experiments) and limited time resolution, which is dictated by the flash-cooling time, which is just below 1 ms in the best case for microcrystals (Clinger et al, 2021). However, they do benefit from three significant advantages: (i) production of diffraction data usually at much higher resolution than at room temperature, (ii) ease of diffraction data analysis (for the single-crystal case) and (iii) potentially permitting the accumulation of higher intermediate-state occupancy.…”
Section: Interest Of Cryo-trapping Studiesmentioning
confidence: 99%
“…Similarly, the drop-on-drop method consists of delivering bursts of picolitre-sized substrate/cofactor drops onto crystals positioned on a moving tape (Butryn et al, 2021). All of these techniques have primarily been developed for roomtemperature time-resolved applications, but they could be adapted for cryo-trapping approaches for timescales above the typical flash-cooling time (�1-100 ms depending on the crystal size) using the MMQX (millisecond mix-and-quench crystallography; Clinger et al, 2021) and spitrobot (Mehrabi et al, 2023) approaches.…”
Section: Examples Of Tr-mx Studies Of Biological Systems Relying On L...mentioning
confidence: 99%
“…Main-chain conformational ensembles may be substantially perturbed, including in ways that may be atypical of crowded cellular environments. Cooling times in cryo-EM (>0.1 ms; Ravelli et al, 2020;Engstrom et al, 2021) are only modestly faster than the $1 ms achievable in cryocrystallography (Clinger et al, 2021), and are likely to be too slow to reliably capture the room/biological temperature conformational ensembles of side-chain rotamers and perhaps also of smaller loops and flaps. Precooling by $30-50 K as cryo-EM samples traverse (in $1-2 ms) the few millimetres of cold gas that is invariably present above liquid ethane (Engstrom et al, 2021) is likely to further perturb side-chain conformations.…”
Section: Complementing Single-particle Cryo-emmentioning
confidence: 99%