TY - JOUR
T1 - Cryo-EM structures from sub-nl volumes using pin-printing and jet vitrification
AU - Ravelli, Raimond B. G.
AU - Nijpels, Frank J. T.
AU - Henderikx, Rene J. M.
AU - Weissenberger, Giulia
AU - Thewessem, Sanne
AU - Gijsbers, Abril
AU - Beulen, Bart W. A. M. M.
AU - Lopez-Iglesias, Carmen
AU - Peters, Peter J.
N1 - Funding Information:
We thank Dr. Fei Sun (Institute of Biophysics, Chinese Academy of Sciences) for providing apoferritin sample, Pascal Huysmans and Paul Kwant (IDEE, Maastricht) for engineering input, Peter Frederik for helpful discussions and Hang Nguyen for critical reading of the paper. Hans Duimel and Hirotoshi Furusho provided technical support from the UM Microscopy Core Lab, Giancarlo Tria helped with initial experiments, Paul van Schayck with the IT infrastructure, and Roger Jeurissen with the theoretical framework. This research received funding from the Netherlands Organization for Scientific Research (NWO) in the framework of the Fund New Chemical Innovations, numbers 731.014.109 and 731.016.407, as well as from the Province of Limburg, the Netherlands.
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/5/22
Y1 - 2020/5/22
N2 - 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 similar to 3 angstrom resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies. There is a need to further improve the automation of cryo-EM sample preparation to make it more easily accessible for non-specialists, reduce sample waste and increase reproducibility. Here, the authors present VitroJet, a single device, where sub-nl volumes of samples are deposited by pin printing thus eliminating the need for sample blotting, which is followed by jet vitrification, and they show that high-resolution structures can be obtained using four standard proteins.
AB - 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 similar to 3 angstrom resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies. There is a need to further improve the automation of cryo-EM sample preparation to make it more easily accessible for non-specialists, reduce sample waste and increase reproducibility. Here, the authors present VitroJet, a single device, where sub-nl volumes of samples are deposited by pin printing thus eliminating the need for sample blotting, which is followed by jet vitrification, and they show that high-resolution structures can be obtained using four standard proteins.
KW - CRYOELECTRON MICROSCOPY
KW - GRID PREPARATION
KW - SPOTITON
KW - FEATURES
KW - SYSTEM
U2 - 10.1038/s41467-020-16392-5
DO - 10.1038/s41467-020-16392-5
M3 - Article
C2 - 32444637
SN - 2041-1723
VL - 11
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 2563
ER -