National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
R01AI147890
United States
Citation
Journal: J Struct Biol / Year: 2022 Title: Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs. Authors: Martin Obr / Wim J H Hagen / Robert A Dick / Lingbo Yu / Abhay Kotecha / Florian K M Schur / Abstract: The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for ...The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.
History
Deposition
Dec 17, 2021
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Header (metadata) release
Jan 12, 2022
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Map release
Jan 12, 2022
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Update
Dec 13, 2023
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Current status
Dec 13, 2023
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
EMPIAR-10889 (Title: Tilt series of EIAV CASPNC VLPs acquired on Krios G4 with Selectris X and Falcon 4 Data size: 461.4 Data #1: Unaligned movies of EIAV VLPs [micrographs - multiframe] Data #2: Tilt series of EIAV VLPs [tilt series])
Model: C-flat-2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: LEICA EM GP Details: 3.5 seconds blotting time, using back-side blotting.
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Electron microscopy
Microscope
TFS KRIOS
Specialist optics
Energy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recording
Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 1.02 sec. / Average electron dose: 3.5 e/Å2 / Details: Images acquired in the EER format
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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