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.
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 -