Netherlands Organisation for Scientific Research (NWO)
NWO.STU.018-2.007
Netherlands
Citation
Journal: Elife / Year: 2022 Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes. Authors: Stefan T Huber / Edin Sarajlic / Roeland Huijink / Felix Weis / Wiel H Evers / Arjen J Jakobi / Abstract: Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness ...Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
EMPIAR-10708 (Title: Apoferritin, TMV and T20S proteasome in nanofluidic channels Data size: 308.3 Data #1: Unaligned multi-frame movies of pyrococcus furiosus apoferritin in silicon nitride nanochannels [micrographs - multiframe] Data #2: Multi-frame movies of T20S proteasome in silicon nitride nanochannels [micrographs - multiframe] Data #3: Multi-frame movies of TMV in silicon nitride nanochannels [micrographs - multiframe])
Cryogen name: ETHANE / Instrument: LEICA PLUNGER Details: The sample was filled into cryoChips through the cantilever and then transferred within ~10 seconds to the Leica plunger for freezing..
Details
The sample was filled into nanofluidic channels.
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Electron microscopy
Microscope
JEOL 3200FSC
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 121 / Average exposure time: 11.0 sec. / Average electron dose: 59.7 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.1 mm
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