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Structure paper

TitleTime-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting.
Journal, issue, pagesNat Methods, Vol. 20, Issue 9, Page 1400-1408, Year 2023
Publish dateAug 17, 2023
AuthorsStefania Torino / Mugdha Dhurandhar / Annelore Stroobants / Raf Claessens / Rouslan G Efremov /
PubMed AbstractSingle-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.
External linksNat Methods / PubMed:37592181
MethodsEM (single particle)
Resolution2.1 - 7.1 Å
Structure data

EMDB-16091, PDB-8bk7:
Cryo-EM structure of beta-galactosidase at 3.3 A resolution plunged 5 ms after mixing with apoferritin
Method: EM (single particle) / Resolution: 3.3 Å

EMDB-16092, PDB-8bk8:
Cryo-EM structure of beta-galactosidase at 2.9 A resolution plunged 205 ms after mixing with apoferritin
Method: EM (single particle) / Resolution: 2.9 Å

EMDB-16093, PDB-8bk9:
Cryo-EM structure of mouse heavy-chain apoferritin at 2.1 A plunged 5ms after mixing with b-galactosidase
Method: EM (single particle) / Resolution: 2.1 Å

EMDB-16094, PDB-8bka:
Cryo-EM structure of mouse heavy-chain apoferritin at 2.7 A plunged 35ms after mixing with b-galactosidase
Method: EM (single particle) / Resolution: 2.7 Å

EMDB-16095, PDB-8bkb:
Cryo-EM structure of mouse heavy-chain apoferritin at 2.2 A plunged 205ms after mixing with b-galactosidase
Method: EM (single particle) / Resolution: 2.2 Å

EMDB-16097, PDB-8bkg:
Cryo-EM structure of beta-galactosidase at 3.2 A resolution plunged 35 ms after mixing with apoferritin
Method: EM (single particle) / Resolution: 3.2 Å

EMDB-16099: GroEL:GroES-ATP complex under continuous turnover condirions
PDB-8bkz: GroEL:GroES-ATP complex under continuous turnover conditions
Method: EM (single particle) / Resolution: 2.3 Å

EMDB-16100, PDB-8bl2:
Structure of GroEL-ATP complex plunge frozen 200 ms after reaction initiation
Method: EM (single particle) / Resolution: 2.3 Å

EMDB-16102, PDB-8bl7:
Structure of GroEL-nucleotide complex in ADP-like conformation plunged 13 ms after mixing with ATP
Method: EM (single particle) / Resolution: 4.4 Å

EMDB-16106, PDB-8blc:
Structure of the GroEL-ATP complex plunge-frozen 50 ms after mixing with ATP
Method: EM (single particle) / Resolution: 2.7 Å

EMDB-16107, PDB-8bld:
Structure of the GroEL(ATP7/ADP7) complex plunged 13 ms after mixing with ATP
Method: EM (single particle) / Resolution: 4.4 Å

EMDB-16108, PDB-8ble:
Structure of GroEL-nucleotide complex in ADP-like conformation plunged 50 ms after mixing with ATP
Method: EM (single particle) / Resolution: 4.0 Å

EMDB-16109, PDB-8blf:
Structure of the GroEL(ATP7/ADP7) complex plunged 50 ms after mixing with ATP
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-16115, PDB-8bly:
Structure of the GroEL-ATP complex plunge-frozen 13 ms after mixing with ATP
Method: EM (single particle) / Resolution: 3.2 Å

EMDB-16116, PDB-8bm0:
Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after reaction initiation
Method: EM (single particle) / Resolution: 3.4 Å

EMDB-16117, PDB-8bm1:
Structure of GroEL:GroES-ATP complex under continuous turnover conditions
Method: EM (single particle) / Resolution: 2.7 Å

EMDB-16118, PDB-8bmd:
Structure of GroEL-ATP complex under continuous turnover conditions
Method: EM (single particle) / Resolution: 2.8 Å

EMDB-16119, PDB-8bmo:
Structure of GroEL:GroES complex exhibiting ADP-conformation in trans ring obtained under the continuous turnover conditions
Method: EM (single particle) / Resolution: 3.4 Å

EMDB-16125, PDB-8bmt:
Structure of GroEL:GroES-ATP complex plunge frozen 200 ms after reaction initiation
Method: EM (single particle) / Resolution: 2.5 Å

EMDB-16154: Map of the GroEL-ES-ATP complex plunge-frozen 50 ms after mixing with ATP
Method: EM (single particle) / Resolution: 7.1 Å

EMDB-16157: Map of GroEL:GroES-(ADP) complex plunge frozen 200 ms after reaction initiation with ATP
Method: EM (single particle) / Resolution: 6.3 Å

Chemicals

ChemComp-FE:
Unknown entry / Iron

ChemComp-HOH:
WATER / Water

ChemComp-MG:
Unknown entry

ChemComp-K:
Unknown entry

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

Source
  • escherichia coli (E. coli)
  • escherichia coli k-12 (bacteria)
  • mus musculus (house mouse)
KeywordsHYDROLASE / Glycosyl hydrolase / METAL BINDING PROTEIN / iron storage / ferritin / octahedral / CHAPERONE / GroEL / GroES

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