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200 kv glacios

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The 200-kV Glacios is an electron microscope designed for high-resolution structural analysis. It operates at an accelerating voltage of 200 kilovolts and utilizes a specialized cryo-electron microscopy (cryo-EM) setup to enable the study of biological samples in their native state.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (6 mentions) K2 summit direct electron detector, by Ametek (2 mentions) Titan krios, by Thermo Fisher Scientific (2 mentions) F416 cmos camera, by TVIPS (1 mentions) Tecnai g2, by Thermo Fisher Scientific (1 mentions) Solarus 2 plasma cleaner, by Ametek (1 mentions) Pelco easiglow glow discharge cleaning system, by Ted Pella (1 mentions) Titan krios g2, by Thermo Fisher Scientific (1 mentions) R1.2 1 3 200 mesh cu, by Quantifoil (1 mentions) K3 direct electron detector, by Ametek (1 mentions) Falcon 4 detector, by Thermo Fisher Scientific (1 mentions) Epu software, by Thermo Fisher Scientific (1 mentions) Falcon 4, by Thermo Fisher Scientific (1 mentions)

7 protocols using 200 kv glacios

1

Cryo-EM and Negative Staining Protocols for Structural Analysis

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Negative staining EM data were collected with an FEI Tecnai G2 transmission electron microscope equipped with a LaB6 emission filament operating at a 200 kV acceleration voltage. Images were captured on a TVIPS F416 CMOS camera at 50,000× magnification and 1.5–2.5 μm underfocus. The corresponding pixel size was 2.13 Å per pixel. A total of 1632 images were acquired.
Cryo-EM data were collected using a Glacios 200 kV (Thermo Fisher) transmission electron microscope equipped with an FEG operating at a 200 kV acceleration voltage. Images were captured using a Falcon 3 direct detection camera at 60,000× magnification and 1–3 μm underfocus. The corresponding pixel size was 2.5 Å per pixel. Each image acquisition was performed in dose-fractionated mode with 60 frames over 2.2 s exposition time for a total dose of 60 e–/Å2 (1 e–/Å2 per frame). A total of 4223 movies were acquired.
Maufront J., Guichard B., Cao L.Y., Cicco A.D., Jégou A., Romet-Lemonne G, & Bertin A. (2022). Direct observation of the conformational states of formin mDia1 at actin filament barbed ends and along the filament. Molecular Biology of the Cell, 34(1), ar2.
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2

Cryo-EM Reveals Particle Morphology

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Cryo-electron microscopy can reveal the diversity of particle size and shape. The morphology of SH-β-CD was examined under Cryo-EM (Glacios-200 KV, Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA, USA).
Yue Y., Fang Y., Jia R., Cao K., Chen X., Xia H, & Cheng Z. (2023). Study on the Antioxidant Effect of Shikonin-Loaded β-Cyclodextrin Forming Host–Guest Complexes That Prevent Skin from Photoaging. International Journal of Molecular Sciences, 24(20), 15177.
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3

SARS-CoV-2 Spike Protein Binding Assay

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For negative stain, purified S protein (0.05 mg/ml) was mixed with soluble ACE2 (0.05 mg/ml) and incubated on ice for 15 min. For the competition experiment, the S protein (0.05 mg/ml) was first incubated on ice with VH ab8 (0.02 mg/ml) for 30 min, followed by addition of ACE2 (0.05 mg/ml) for another 30 min. Grids (copper 200 or 300 mesh coated with continuous ultrathin carbon) were plasma cleaned using an H2/O2 gas mixture for 15 s in a Solarus II Plasma Cleaner (Gatan) or 10 s in a PELCO easiGlow Glow Discharge Cleaning System (Ted Pella). The protein mixtures (4.8 μl) were applied to the grid and allowed to adsorb for 30 s before blotting away excess liquid, followed by a brief wash with Milli-Q H2O. Grids were stained by 3 successive applications of 2% (w/v) uranyl formate (20 s, 20 s, 60 s). Negative stain grids were imaged using a 200-kV Glacios (Thermo Fisher Scientific) transmission electron microscope (TEM) equipped with a Falcon3 camera operated in linear mode. Micrographs were collected using EPU at nominal 92,000× magnification (physical pixel size 1.6 Å) over a defocus range of −2.0 μm to −1.0 μm with a total accumulated dose of 40 e–/Å2.
Zhu X., Mannar D., Srivastava S.S., Berezuk A.M., Demers J.P., Saville J.W., Leopold K., Li W., Dimitrov D.S., Tuttle K.S., Zhou S., Chittori S, & Subramaniam S. (2021). Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies. PLoS Biology, 19(4), e3001237.
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4

Cryo-EM Data Acquisition and Processing

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The frozen-hydrated samples were loaded on the 200-kV Glacios (Thermo Fisher Scientific) with a K2 Summit direct electron detector (Gatan, Inc.). The micrographs were collected in super-resolution mode operated using SerialEM (Version 3) software (70 (link)). A total of 731 movies were acquired at a nominal magnification of 36,000x with a physical pixel size of 1.143 Å on the specimen level. The movies were dose fractionated into 40 frames of 0.25 s each at a dose rate of 5.8 electrons/Å2/s for a total dose of 58 e-/ Å2 over a defocus range between −0.9 µm and −2.0 µm. The image processing was done using RELION 3.1 (47 ). All the 731 micrographs were motion-corrected and dose-weighted using MotionCor2 (71 (link)) with a binning factor of 2 and divided into 5 × 5 patches. The contrast transfer function was calculated with CTFFIND-4.1 (72 (link)). A template-free auto-picking procedure based on Laplacian-of-Gaussian (LoG) filter was used for auto-picking, which yielded a dataset of 635,143 particles. Several rounds of 2D classifications using a mask of 70 Å diameter were carried out to remove bad particles and obtain a final set of homogenous particles.
Bera M., Radhakrishnan A., Coleman J., Sundaram R.V., Ramakrishnan S., Pincet F, & Rothman J.E. (2023). Synaptophysin Chaperones the Assembly of 12 SNAREpins under each Ready-Release Vesicle. bioRxiv.
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5

Cryo-EM Sample Preparation for NR5A2–Nucleosome

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To prepare the cryo-EM specimen, the sample (4 μl) was applied to a glow-discharged holey carbon grid (Quantifoil R1.2/1.3 200-mesh Cu). The grids were blotted for 3.0 s at a blotting strength setting of 5 under 100% humidity at 4 °C and then plunged into liquid ethane and cooled by liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher). Data acquisition for the NR5A2–nucleosome complexSHL+5.5 was conducted using a 300 kV Titan Krios G2 (Thermo Fisher Scientific) equipped with a GIF Quantum 967 energy filter and a K3 direct electron detector (Gatan) running in correlated double sampling mode at a magnification factor of 105kx, equivalent to a pixel size of 0.85 Å per pixel. Automated data acquisition was performed using SerialEM software. A total of 12,510 videos were collected, with a total electron dose of 65.4 e/Ų fractionated over 40 frames. The data set for pure nucleosomes was acquired on a 200 kV Glacios (Thermo Fisher Scientific) equipped with a K2 Summit direct electron detector (Gatan) at a magnification factor of 22kx, equivalent to a pixel size of 1.89 Å per pixel. Here, 1,998 videos were collected, with a total electron dose of 61 e/Ų fractionated over 40 frames.
Kobayashi W., Sappler A.H., Bollschweiler D., Kümmecke M., Basquin J., Arslantas E.N., Ruangroengkulrith S., Hornberger R., Duderstadt K, & Tachibana K. (2024). Nucleosome-bound NR5A2 structure reveals pioneer factor mechanism by DNA minor groove anchor competition. Nature Structural & Molecular Biology, 31(5), 757-766.
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6

Cryo-EM Imaging of Virions

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2.5 μL of virions, measured at a PFU of 1 × 1013 phages/mL, was applied to a 200-mesh copper Quantifoil R 2/1 holey carbon grid (EMS) previously glow-discharged for 60 sec at 15 mA using an easiGlow (PELCO). The grid was blotted for 7.5 sec at blot force 2 and vitrified immediately in liquid ethane using a Vitrobot Mark IV (Thermo Scientific). Cryo-grids were screened on 200 kV Glacios (Thermo Scientific) equipped with a Falcon4 detector (Thermo Scientific) at Thomas Jefferson University. EPU software (Thermo Scientific) was used for data collection using accurate positioning mode. For high-resolution data collection of the Pa193, micrographs were collected on a Titan Krios (Thermo Scientific) microscope operated at 300 kV and equipped with a K3 direct electron detector camera (Gatan) at the National Cryo-EM Facility at the Pacific Northwest Cryo-EM Center, (PNCC).
Cingolani G., Iglesias S., Hou C.F., Lemire S., Soriaga A, & Kyme P. (2024). Cryo-EM analysis of Pseudomonas phage Pa193 structural components. Research Square.
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7

Cryo-EM Preparation of Protein Complexes

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For all complexes, 0.05% (w/v) β-OG (n-octyl-β-d-glucopyranoside) was added to the sample immediately before plunge freezing. Three microliters of sample was applied to r2/2 Quantifoil mesh 300 grids, and after 20 s of incubation, the excess sample was blotted away and grids were plunge-frozen in liquid ethane [blot force of −10, blot time of 2 s, 4°C, 100% humidity, Vitrobot Mark IV (Thermo Fisher Scientific)]. The grids were screened on a 200 kV Glacios (Thermo Fisher Scientific), and movies were recorded on a 300 kV Titan Krios (Thermo Fisher Scientific) with a Falcon IV (Thermo Fisher Scientific) or K3 (Gatan) direct electron detector [Electron Bio-imaging Centre (eBIC) and Medical Research Council Laboratory of Molecular Biology (MRC-LMB)]. Data collection parameters and metrics are listed in table S2.
Dendooven T., Zhang Z., Yang J., McLaughlin S.H., Schwab J., Scheres S.H., Yatskevich S, & Barford D. (2023). Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere. Science Advances, 9(30), eadg7480.
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