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Title | Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting. |
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Journal, issue, pages | Nat Methods, Vol. 20, Issue 9, Page 1400-1408, Year 2023 |
Publish date | Aug 17, 2023 |
![]() | Stefania Torino / Mugdha Dhurandhar / Annelore Stroobants / Raf Claessens / Rouslan G Efremov / ![]() |
PubMed Abstract | Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient ...Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex. |
![]() | ![]() ![]() |
Methods | EM (single particle) |
Resolution | 2.1 - 7.1 Å |
Structure data | EMDB-16091, PDB-8bk7: EMDB-16092, PDB-8bk8: EMDB-16093, PDB-8bk9: EMDB-16094, PDB-8bka: EMDB-16095, PDB-8bkb: EMDB-16097, PDB-8bkg: EMDB-16099: GroEL:GroES-ATP complex under continuous turnover condirions EMDB-16100, PDB-8bl2: EMDB-16102, PDB-8bl7: EMDB-16106, PDB-8blc: EMDB-16107, PDB-8bld: EMDB-16108, PDB-8ble: EMDB-16109, PDB-8blf: EMDB-16115, PDB-8bly: EMDB-16116, PDB-8bm0: EMDB-16117, PDB-8bm1: EMDB-16118, PDB-8bmd: EMDB-16119, PDB-8bmo: EMDB-16125, PDB-8bmt: ![]() EMDB-16154: Map of the GroEL-ES-ATP complex plunge-frozen 50 ms after mixing with ATP ![]() EMDB-16157: Map of GroEL:GroES-(ADP) complex plunge frozen 200 ms after reaction initiation with ATP |
Chemicals | ![]() ChemComp-FE: ![]() ChemComp-HOH: ![]() ChemComp-MG: ![]() ChemComp-K: ![]() ChemComp-ATP: |
Source |
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![]() | HYDROLASE / Glycosyl hydrolase / METAL BINDING PROTEIN / iron storage / ferritin / octahedral / CHAPERONE / GroEL / GroES |