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Titan krios g2

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The Titan Krios G2 is a high-resolution cryo-electron microscope designed for advanced structural biology research. It features a stable and high-performance electron optical system, enabling the analysis of complex biological macromolecules at near-atomic resolution.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (18 mentions) K3 detector, by Ametek (2 mentions) K2 summit direct electron detector, by Ametek (2 mentions) R1.2 1 3 200 mesh cu, by Quantifoil (2 mentions) Falcon 4i, by Thermo Fisher Scientific (1 mentions) Falcon 4, by Thermo Fisher Scientific (1 mentions) Krios g3i microscope, by Thermo Fisher Scientific (1 mentions) Glacios microscope, by Thermo Fisher Scientific (1 mentions) Epu version 2, by Thermo Fisher Scientific (1 mentions) Plasma cleaner pdc 002 ce, by Harrick (1 mentions) Vitrobot filter paper grade 595, by Ted Pella (1 mentions) Quantum energy filter, by Thermo Fisher Scientific (1 mentions) K2 summit direct detector, by Ametek (1 mentions) Talos f200c, by Thermo Fisher Scientific (1 mentions) Ceta 16m camera, by Thermo Fisher Scientific (1 mentions) Gif quantum ls energy filter, by Ametek (1 mentions) 200 kv glacios, by Thermo Fisher Scientific (1 mentions) K3 direct electron detector, by Ametek (1 mentions) Digital micrograph software, by Ametek (1 mentions) K2 summit camera, by Thermo Fisher Scientific (1 mentions)

21 protocols using titan krios g2

1

Cryo-EM Data Acquisition and Processing

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For sample-specific details, refer to Table S6.
Movies were collected in compressed tiff format on a Titan Krios G2 (Thermo Fisher) operating at 300 kV with a K3 detector (Gatan) in super resolution counting mode using a custom version of EPU 2.5 (Thermo Fisher). A defocus range of 0.8-2.6 μm was applied with a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel and with a total dose of 43-47 e/ Å2, see Table S6.
Two-times binned movies were then motion corrected and aligned on the fly using Relion(3.1) scheduler (Zivanov et al., 2018 (link)) with a 5 × 5 patch based alignment. CTF-estimation of full-frame non-weighted micrographs was performed with the GCTF (1.06) (Zhang, 2016 (link)) module in cryoSPARC(v2.14.1-live) (Punjani et al., 2017 (link)).
Dejnirattisai W., Zhou D., Ginn H.M., Duyvesteyn H.M., Supasa P., Case J.B., Zhao Y., Walter T.S., Mentzer A.J., Liu C., Wang B., Paesen G.C., Slon-Campos J., López-Camacho C., Kafai N.M., Bailey A.L., Chen R.E., Ying B., Thompson C., Bolton J., Fyfe A., Gupta S., Tan T.K., Gilbert-Jaramillo J., James W., Knight M., Carroll M.W., Skelly D., Dold C., Peng Y., Levin R., Dong T., Pollard A.J., Knight J.C., Klenerman P., Temperton N., Hall D.R., Williams M.A., Paterson N.G., Bertram F.K., Siebert C.A., Clare D.K., Howe A., Radecke J., Song Y., Townsend A.R., Huang K.Y., Fry E.E., Mongkolsapaya J., Diamond M.S., Ren J., Stuart D.I, & Screaton G.R. (2021). The antigenic anatomy of SARS-CoV-2 receptor binding domain. Cell, 184(8), 2183-2200.e22.
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2

Cryo-EM Data Acquisition Workflow

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Single-particle cryo-EM data were collected on the Thermo Fisher Scientific Titan Krios G2 transmission electron microscope with a Thermo Fisher Scientific Falcon 4i direct electron detector and SelectrisX energy filter. Data were collected with an accelerating voltage of 300 kV and nominal magnification of ×165,000, which corresponds to a pixel size of 0.74 Å (full data acquisition settings are shown in Extended Data Table 1). A total of 59,394 cryo-EM videos was acquired.
Makhlouf L., Peter J.J., Magnussen H.M., Thakur R., Millrine D., Minshull T.C., Harrison G., Varghese J., Lamoliatte F., Foglizzo M., Macartney T., Calabrese A.N., Zeqiraj E, & Kulathu Y. (2024). The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons. Nature, 627(8003), 437-444.
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3

Cryo-EM Structure Determination Protocol

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Cryo-EM grids were prepared as described above with 1.5 mg ml−1 sample. Single-particle cryo-EM data were collected on the Thermo Fisher Scientific Titan Krios G2 transmission electron microscope with a Thermo Fisher Scientific Falcon 4 direct electron detector. Data were collected with an accelerating voltage of 300 kV and a nominal magnification of ×96,000, corresponding to a pixel size of 0.82 Å (full data-acquisition settings shown in Extended Data Table 1). A total of 3,028 cryo-EM videos was acquired. The data were processed as previously, with the final map being generated from the particles after several rounds of 3D variability analysis.
Makhlouf L., Peter J.J., Magnussen H.M., Thakur R., Millrine D., Minshull T.C., Harrison G., Varghese J., Lamoliatte F., Foglizzo M., Macartney T., Calabrese A.N., Zeqiraj E, & Kulathu Y. (2024). The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons. Nature, 627(8003), 437-444.
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4

Graphene Oxide Grid Preparation for Electron Microscopy

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Graphene oxide (GO) grids were prepared following a published
procedure31 using as
support Quantifoil Cu/Rh 200 mesh R2/2 grids. 3 μL of TG sample was
applied to the GO grids at a concentration of approximately 0.05 mg/mL and
plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). While
eTG behaved better when deglycosylated, there was no difference between
glycosylated and deglycosylated rTG. Therefore, we collected datasets of
deglycosylated eTG and non-deglycosylated rTG.
Images were acquired on a K2 Summit detector (Gatan) in counting mode
using a Titan Krios G2 (Thermo Fisher) electron microscope at 300 kV. A Quantum
GIF energy filter (Gatan) was used with a slit width of 20 eV to remove
inelastically scattered electrons. The eTG dataset was collected at eBIC
(Diamond Light Source, UK) while the rTG dataset was collected at MRC-LMB. For
the eTG dataset, 40 movie frames were recorded, using a fluency of 1.18
electrons per Å2 per frame, for a total accumulated dose of
47.2 electrons per Å2 at a pixel size of 1.043 Å on the
specimen. For the rTG dataset 52 movie frames were recorded, using a fluency of
0.91 electrons per Å2 per frame, for a total accumulated dose
of 36.3 electrons per Å2 at a pixel size of 1.149 Å on
the specimen. Further details are presented in Extended Data Table 1.
Coscia F., Taler-Verčič A., Chang V.T., Sinn L., O'Reilly F.J., Izoré T., Renko M., Berger I., Rappsilber J., Turk D, & Löwe J. (2020). The structure of human thyroglobulin. Nature, 578(7796), 627-630.
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5

Cryo-EM Data Acquisition Using Titan Krios

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For each cryo-EM sample, a dataset was recorded in Energy-Filtered Transmission Electron Microscopy (EF-TEM) mode using either a Titan Krios G2 or a Krios G3i microscope (Thermo Scientific), both operated at 300 kV. Electron-optical alignments were adjusted with EPU 2.9–2.11 (Thermo Scientific). Images were recorded using automation strategies of EPU 2.9–2.11 in electron counting mode with either a Gatan K2 (installed on Krios G2) or a Gatan K3 (installed on Krios G3i) direct electron detector at a nominal magnification of 105,000, corresponding to a calibrated pixel size of 0.831 and 0.837 Å, respectively. Dose fractionated movies (40 frames) were recorded at an electron flux of approximately 15 e− pixel−1 s−1 for 2 s, corresponding to a total dose of roughly 40 e−/A2. Images were recorded between −1.1 and −2.1 µm nominal defocus. CryoSPARC Live v.3.0 was used for real-time cryo-EM data quality assessment.
Wu D., Mehdipour A.R., Finke F., Goojani H.G., Groh R.R., Grund T.N., Reichhart T.M., Zimmermann R., Welsch S., Bald D., Shepherd M., Hummer G, & Safarian S. (2023). Dissecting the conformational complexity and mechanism of a bacterial heme transporter. Nature Chemical Biology, 19(8), 992-1003.
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6

Cryo-EM Structural Analysis of SARS-CoV-2 Spike-CR3022 Fab

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Purified spike protein was buffer exchanged into 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3 buffer using a desalting column (Zeba, Thermo Fisher). A final concentration of 0.2 mg/mL was incubated with CR3022 Fab (in the same buffer) in a 6:1 molar ratio (Fab to trimeric spike) at room temperature. Aliquots were taken at 50 min and 3 h and 3 μL immediately applied to a holey carbon-coated 200 mesh copper grid (C-Flat, CF-2/1, Protochips) that had been freshly glow discharged on high for 20 s (Plasma Cleaner PDC-002-CE, Harrick Plasma) and excess liquid removed by blotting for 6 s with a blotting force of −1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100% relative humidity. Blotted grids were then immediately plunge frozen using a Vitrobot Mark IV (Thermo Fisher).
Frozen grids were first screened on a Glacios microscope operating at 200 kV (Thermo Fisher) before imaging on a Titan Krios G2 (Thermo Fisher) at 300 kV. Movies (40 frames each) were collected in compressed tiff format on a K3 detector (Gatan) in super resolution counting mode using a custom EPU version 2.5 (Thermo Fisher) with a defocus range of 0.8-2.6 μm and at a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel, see Table S4.
Huo J., Zhao Y., Ren J., Zhou D., Duyvesteyn H.M., Ginn H.M., Carrique L., Malinauskas T., Ruza R.R., Shah P.N., Tan T.K., Rijal P., Coombes N., Bewley K.R., Tree J.A., Radecke J., Paterson N.G., Supasa P., Mongkolsapaya J., Screaton G.R., Carroll M., Townsend A., Fry E.E., Owens R.J, & Stuart D.I. (2020). Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike. Cell Host & Microbe, 28(3), 445-454.e6.
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7

Cryo-ET of Ce-lamin Filaments

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A 3 μl drop of assembled Ce-lamins was applied onto a glow-discharged 200-mesh carboncoated copper grid (Quantifoil, Jenna, Germany), which was coated with an additional layer of ~2 nm carbon, and vitrified immediately. For cryo-ET, fiducial 10 nm BSA gold tracer markers (Aurion) were added prior to vitrification. Data was collected using a Titan Krios G2 (Thermo Scientific) electron microscope equipped with a Gatan Quantum Energy Filter with a K2 or K3 direct electron detector. Images were acquired in counting mode at a magnification of 81.000 x, resulting in a pixel size of 1.72 Å2. Images of Ce-lamin Δcoil 1A filaments were collected with a pixel size of 81.000 x, resulting in a pixel size of 1.062 Å2, using a K3 detector.
2997 images were collected with a defocus range between 1.8 and 2.8 µm underfocus. Tomograms were acquired using the SerialEM software package [27 (link)] in a bidirectional fashion, starting at −30°. Tilt series were collected between −60° and 60°, with 3° interval at a defocus of −4 µm. Each tilt movie was exposed for 1.4 s in 0.2 s frames; with 1.95 e−/Å2 per tilt and a total dose of 112 e−/Å2. Tilt series were collected with a flux of 9.6 e−/pixel/s, at a magnification of 64.000 x and a corresponding pixel size of 2.22 Å2.
de Leeuw R., Kronenberg-Tenga R., Eibauer M, & Medalia O. (2022). Filament assembly of the C. elegans lamin in the absence of helix 1A. Nucleus, 13(1), 49-57.
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8

CryoEM Imaging of Nucleosome Complexes

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CryoEM data were collected using a Titan Krios G2 (Thermo Fisher Scientific) operated at 300keV, with a BioQuantum energy filter equipped with a K2 Summit direct detector (Gatan, Inc. Pleasanton, CA). Movies were acquired in energy filtered mode with an energy slit of 20eV, magnification of ~165kx (pixel size of 0.849 angstroms/pixel). The electron beam had a flux rate of 4.9 electrons/(angstrom)2/s, and movies were acquired at 4 frames/s for duration of 10 seconds, for a total electron interaction of 49 electrons/(angstrom)2. A 70μm condenser aperture and a 100μm objective aperture were used while imaging. Data were collected using SerialEM automated acquisition software (Mastronarde, 2005 (link)). Micrographs for the direct comparison between CEN-NCP and H3-NCP in the presence or absence of Tween 20 were collected on a Talos F200C (Thermo Fisher Scientific) operated at 200keV with a Ceta 16M camera (Thermo Fisher Scientific).
Migl D., Kschonsak M., Arthur C.P., Khin Y., Harrison S.C., Ciferri C, & Dimitrova Y.N. (2020). Cryo-EM Structure of a Yeast Centromeric Nucleosome at 2.7 Å resolution. Structure (London, England : 1993), 28(3), 363-370.e3.
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9

Cryo-EM Structure of CaV2.3 Complex

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Holey carbon grids (Au R1.2/1.3 300 mesh) (Quantifoil Micro Tools, Germany) were glow-discharged using H2 and O2 for 60 s before being loaded with 2.5 μL purified CaV2.3 complex. The grids were automatically blotted for 4 s at 4 °C and 100% humidity and flash-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, USA). Cryo-EM data were collected using a 300 kV Titan Krios G2 (Thermo Fisher Scientific, USA) equipped with a K2 Summit direct electron detector (Gatan, USA) and a GIF Quantum LS energy filter (Gatan, USA). The dose rate was set to ~9.2 e−/(pixel*s), and the energy filter slit width was set to 20 eV. A total exposure time of 6.72 s was dose-fractioned into 32 frames. A nominal magnification of ×130,000 was used, resulting in a calibrated super-resolution pixel size of 0.52 Å on images. SerialEM49 (link) was used to automatically acquire the movie stacks. The nominal defocus range was set from –1.2 μm to –2.2 μm.
Gao Y., Xu S., Cui X., Xu H., Qiu Y., Wei Y., Dong Y., Zhu B., Peng C., Liu S., Zhang X.C., Sun J., Huang Z, & Zhao Y. (2023). Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3. Nature Communications, 14, 516.
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10

Graphene Oxide Grid Preparation for Electron Microscopy

Check if the same lab product or an alternative is used in the 5 most similar protocols
Graphene oxide (GO) grids were prepared following a published
procedure31 using as
support Quantifoil Cu/Rh 200 mesh R2/2 grids. 3 μL of TG sample was
applied to the GO grids at a concentration of approximately 0.05 mg/mL and
plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher). While
eTG behaved better when deglycosylated, there was no difference between
glycosylated and deglycosylated rTG. Therefore, we collected datasets of
deglycosylated eTG and non-deglycosylated rTG.
Images were acquired on a K2 Summit detector (Gatan) in counting mode
using a Titan Krios G2 (Thermo Fisher) electron microscope at 300 kV. A Quantum
GIF energy filter (Gatan) was used with a slit width of 20 eV to remove
inelastically scattered electrons. The eTG dataset was collected at eBIC
(Diamond Light Source, UK) while the rTG dataset was collected at MRC-LMB. For
the eTG dataset, 40 movie frames were recorded, using a fluency of 1.18
electrons per Å2 per frame, for a total accumulated dose of
47.2 electrons per Å2 at a pixel size of 1.043 Å on the
specimen. For the rTG dataset 52 movie frames were recorded, using a fluency of
0.91 electrons per Å2 per frame, for a total accumulated dose
of 36.3 electrons per Å2 at a pixel size of 1.149 Å on
the specimen. Further details are presented in Extended Data Table 1.
Coscia F., Taler-Verčič A., Chang V.T., Sinn L., O'Reilly F.J., Izoré T., Renko M., Berger I., Rappsilber J., Turk D, & Löwe J. (2020). The structure of human thyroglobulin. Nature, 578(7796), 627-630.
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