National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM120553-01
United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
R01AI070771
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
1R01GM118396-01
United States
Netherlands Organization for Scientific Research
NWO, Rubicon 019.2015.2.310.006
Netherlands
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
T32GM008268
United States
EMBO
ALTF 933-2015
United States
Citation
Journal: J Struct Biol / Year: 2017 Title: Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids. Authors: Joost Snijder / Andrew J Borst / Annie Dosey / Alexandra C Walls / Anika Burrell / Vijay S Reddy / Justin M Kollman / David Veesler / Abstract: Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain ...Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.
History
Deposition
Feb 23, 2017
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Header (metadata) release
Mar 15, 2017
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Map release
Mar 15, 2017
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Update
Jan 29, 2020
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Current status
Jan 29, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK I
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Electron microscopy
Microscope
FEI TECNAI F20
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 100 / Average exposure time: 10.0 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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