Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification

Nat Commun. 2020 May 22;11(1):2563. doi: 10.1038/s41467-020-16392-5.

Abstract

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy
  • Printing, Three-Dimensional* / instrumentation
  • Proteins / chemistry
  • Proteins / ultrastructure*
  • Reproducibility of Results
  • Single Molecule Imaging
  • Specimen Handling / instrumentation
  • Specimen Handling / methods*

Substances

  • Proteins