Titan krios g3i

Manufactured by Thermo Fisher Scientific

Sourced in United States, Poland

The Titan Krios G3i is a high-resolution cryo-electron microscope designed for advanced structural biology research. It features a stable, high-performance electron beam and advanced imaging capabilities to enable researchers to obtain detailed three-dimensional structures of biological macromolecules and complexes.

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Lab products found in correlation

Vitrobot mark 4, by Thermo Fisher Scientific (6 mentions) K3 camera, by Ametek (5 mentions) K3 direct electron detector, by Ametek (4 mentions) Epu software, by Thermo Fisher Scientific (3 mentions) Vitrobot system, by Thermo Fisher Scientific (2 mentions) Talos arctica g2, by Thermo Fisher Scientific (2 mentions) Falcon 3ec camera, by Thermo Fisher Scientific (2 mentions) Falcon 3ec direct electron detector, by Thermo Fisher Scientific (2 mentions) Vitrobot, by Thermo Fisher Scientific (2 mentions) Waffle pattern grating replica, by Ted Pella (2 mentions) Bioquantum energy filter, by Ametek (2 mentions) Quantum ls energy filter, by Ametek (1 mentions) K3 summit, by Ametek (1 mentions) Microscopy suite software, by Ametek (1 mentions) Tecnai g2 f20, by Thermo Fisher Scientific (1 mentions) K3 summit direct electron detector, by Ametek (1 mentions) K3 bioquantum detector, by Ametek (1 mentions) Vitrobot mk 4, by Thermo Fisher Scientific (1 mentions) Tecnai spirit tem, by Thermo Fisher Scientific (1 mentions) Em gp2, by Leica (1 mentions)

Spelling variants of this product

Titan Krios (617 citations) Titan Krios G3i microscope (39 citations) Titan Krios G3i electron microscope (16 citations) Titan Krios G3i TEM (5 citations) Titan Krios G3i transmission (4 citations) Krios G3i (3 citations) Titan Krios G3i electron cryo-microscope (2 citations)

31 protocols using titan krios g3i

Cryo-EM Sample Preparation and Imaging

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The concentrated sample (3.5 μL) at a concentration of 12 mg/mL was applied to glow-discharged holey carbon-coated grids (Quantifoil 200 mesh, Au R1.2/1.3). The grids were blotted for 3.5 s and flash-frozen in liquid ethane using a Vitrobot (Mark IV, Thermo Fisher Scientific). Images were recorded on a 300 kV Titan Krios G3i electron microscope (Thermo Fisher Scientific) equipped with Gatan K3 Summit direct detector and a GIF Quantum energy filter (slit width 20 eV). Movie stacks were collected using SerialEM [48 ] in counting mode at a magnification of 105,000× with the corresponding pixel size of 0.85 Å. Movies stacked with 50 frames were exposed for 2 s. Two data sets of the same sample were collected, including 3,447 and 2,799 movies separately. Two data sets were recorded at a total dose of about 56.15 e/Å2 and 58.32 e/Å2, respectively. The defocus range was set from −1.0 μM to −2.0 μM.

Gan B., Yu L., Yang H., Jiao H., Pang B., Chen Y., Wang C., Lv R., Hu H., Cao Z, & Ren R. (2023). Mechanism of agonist-induced activation of the human itch receptor MRGPRX1. PLOS Biology, 21(6), e3001975.

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Cryo-EM Data Acquisition Protocol

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The datasets were collected on a Titan Krios G3i (Thermo Fisher Scientific, USA) 300 kV electron microscope equipped with a GIF Quantum energy filter and a K3 direct electron detector (Gatan, USA). Movies were acquired with homemade scripts in SerialEM^{43 (link)} employing a 9-hole beam-image shift acquisition pattern with 1 image in the center of each hole and saved as non-gain-normalized compressed TIFF files. The acquisition parameters for each dataset are listed in Table 1 and the dataset details in Table 2.

Danev R., Belousoff M., Liang Y.L., Zhang X., Eisenstein F., Wootten D, & Sexton P.M. (2021). Routine sub-2.5 Å cryo-EM structure determination of GPCRs. Nature Communications, 12, 4333.

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Cryo-EM Data Collection and Processing

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Except for the 5HT-B and 6HBC-2 datasets, all the data were acquired at 300 keV on a Titan Krios G3i instrument (Thermo Fisher Scientific) equipped with a K3 camera (Gatan/Ametek) and energy filter operated in the energy-filtered transmission electron microscopy mode using a slit width of 20 eV. The 6HBC-2 and 5HT-B datasets were acquired on Titan Krios G1 equipped with Cs corrector and K2 camera (Gatan/Ametek). The data were collected over a defocus range of −0.5 to −2.0 μm with a targeted dose of 60 e⁻ Å^–2. Automated data collection was performed with EPU and the data saved as gain-normalized compressed TIFF files (K3) or MRC files (K2) with pixel sizes of 0.645 and 0.860 Å px^–1, respectively.

McRae E.K., Rasmussen H.Ø., Liu J., Bøggild A., Nguyen M.T., Sampedro Vallina N., Boesen T., Pedersen J.S., Ren G., Geary C, & Andersen E.S. (2023). Structure, folding and flexibility of co-transcriptional RNA origami. Nature nanotechnology, 18(7), 808-817.

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Structural Characterization of S. enterica WbaP

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Data were collected at the MIT Characterization.nano facility. Optimal freezing conditions were screened using the Vitrobot system (Thermo Scientific). Grids were clipped and screened on a Talos Arctica G2 (Thermo Scientific) equipped with a Falcon 3EC camera (Thermo Scientific). Optimal particles were observed in grid GD5-3, containing 2 mg/mL S. enterica WbaP. Based on these preliminary results, 4,481 movies of SMA30-solubilized S. enterica WbaP were collected on a Titan Krios G3i (Thermo Scientific, Table S1) equipped with a K3 camera (Gatan, Inc). Motion correction, CTF estimation, particle extraction, ab-initio reconstruction, and all refinement were conducted using Cryosparc V4.^{66 ,67 (link)}

Dodge G.J., Anderson A.J., He Y., Liu W., Viner R, & Imperiali B. (2023). Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle. bioRxiv.

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Cryo-EM Imaging of Membrane Protein Complexes

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Grids were screened for ice quality, and cryo-EM data were acquired using a Titan Krios G3i cryo-EM (Thermo Fisher Scientific, Waltham, MA) running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (slit width 25 eV) and a Gatan K3 Summit direct electron detector in the electron counting mode. The electron flux was set to 14 e⁻/pix/s at the detector. For the WT close state with EGTA with EGTA, imaging was performed at a nominal magnification of ×81,000, corresponding to a calibrated pixel size of 1.07 Å/pix (University of Tokyo, Japan). Electron dose was set to 50 e⁻/Å² for the WT close state with EGTA. For other samples, imaging was performed at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83 Å/pix. The electron dose was set to 60 e⁻/Å² for the WT open state and 50 e−/Å2 for K4593A. Each movie was subdivided into 1 e⁻/frame for all datasets. The data were automatically acquired by the image shift method using SerialEM software^{40 (link)}, with a defocus range of −0.8 to −1.6 μm.

Kobayashi T., Tsutsumi A., Kurebayashi N., Saito K., Kodama M., Sakurai T., Kikkawa M., Murayama T, & Ogawa H. (2022). Molecular basis for gating of cardiac ryanodine receptor explains the mechanisms for gain- and loss-of function mutations. Nature Communications, 13, 2821.

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Cryo-Electron Microscopy Imaging Workflow

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After live-cell FLM, cryo-FLM imaging and/or cryo-FIB milling, the same clipped frozen grids were imaged using a Titan Krios G3i (Thermo Fisher Scientific) at 300 kV without the fringe-free optical state. Images were acquired on a Gatan Bioquantum GIF-K3 camera (Gatan) in EFTEM mode using a 20-eV slit. Images were captured at various magnifications of ×81 (4,485 Å/pixel) for whole-grid mosaic collection, ×470 (399 Å per pixel) and ×1,950 (177.6 Å per pixel) for square or whole lamella overview, ×6,500 (27.4 Å per pixel) for intermediate magnification imaging where the FOV is suitable for reliable tracking and 40-nm nanoparticles are visibly distinguished, and ×19,500 (4.603 Å per pixel) for data acquisition using the SerialEM software (v.3.8.7) package^{23 (link)}. Fresh correlated-double sampling mode (CDS) counting gains with the designated beam size (for example, 3.15 μm) were prepared in the Gatan Microscopy Suite Software. The full frame size of 5,760 × 4,092 pixels (counting, CDS mode at the dose rate of 10 e⁻pixel⁻¹s⁻¹, bin2 at hardware in SerialEM) was collected.

Yang J.E., Larson M.R., Sibert B.S., Kim J.Y., Parrell D., Sanchez J.C., Pappas V., Kumar A., Cai K., Thompson K, & Wright E.R. (2023). Correlative montage parallel array cryo-tomography for in situ structural cell biology. Nature Methods, 20(10), 1537-1543.

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Cryo-EM Imaging Protocols for Structural Studies

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Imaging was performed at liquid nitrogen
temperature on a Tecnai T12 instrument (Twin, Thermo Fisher Scientific)
operating at 120 kV, Tecnai G2 F20 instrument (Twin, Thermo Fisher
Scientific) operating at 200 kV, Talos Arctica G3 instrument (Thermo
Fisher Scientific) operating at 200 kV, and on a Titan Krios G3i instrument
(Thermo Fisher Scientific) operating at 300 kV. Specimens were introduced
into the T12 and F20 microscopes using a Gatan 626 cooling holder
and transfer station, or Autoloader for the Arctica and Titan instruments,
and were equilibrated at −178 °C prior to imaging. Images
were recorded using (1) a TVIPS F244HD CCD digital camera or Gatan
OneView CMOS camera, on the Technai T12 instrument; (2) a Gatan US4000
CCD digital instrument; (3) a Gatan OneView CMOS camera on the Talos
Arctica; and (4) a Falcon 3EC direct electron detector (Thermo Fisher
Scientific), using the electron counting mode. A Volta phase plate
was applied in specified cases on the Titan Krios instrument. Analysis
and processing of the movies and images were done using Digital Micrograph,
GMS 3, imageJ, and irfanview. The samples were imaged at various magnifications,
using total electron doses not exceeding 20 e/Å².

Tsarfati Y., Biran I., Wiedenbeck E., Houben L., Cölfen H, & Rybtchinski B. (2021). Continuum Crystallization Model Derived from Pharmaceutical Crystallization Mechanisms. ACS Central Science, 7(5), 900-908.

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Cryo-EM of hXKR8–hBSGΔ-FLAG–Fab Complex

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In cryo-EM, fluorinated Fos-choline 8 was added at a final concentration of 0.075% to the hXKR8–hBSGΔ-FLAG–Fab complex in lipid nanodiscs. An aliquot (3 μl) of the sample was applied to a glow-discharged Quantifoil holey carbon grid (Au, R1.2/1.3, 300-mesh), blotted for 4 s (+10 blot force, 10 s wait time, and 100% humidity at 6 °C), and plunge-frozen by Vitrobot (Thermo Fisher Scientific). Movies were acquired using a Titan Krios G3i (Thermo Fisher Scientific) equipped with a K3 summit direct electron detector (Gatan) running at 300 kV operated in the counting mode with a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83 Å. Data were automatically acquired using EPU software (Thermo Fisher Scientific). The dose rate was 10.432 e⁻/Å² per second, and the total exposure time was 4.79 s, resulting in a total dose of 50 e⁻/Å² over 50 frames. A total of 6118 movies were collected.

Sakuragi T., Kanai R., Otani M., Kikkawa M., Toyoshima C, & Nagata S. (2024). The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase. The Journal of Biological Chemistry, 300(3), 105755.

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Cryo-EM Analysis of S. enterica WbaP

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Data were collected at the MIT Characterization.nano facility. Optimal freezing conditions were screened using the Vitrobot system (Thermo Fisher Scientific). Grids were clipped and screened on a Talos Arctica G2 (Thermo Fisher Scientific) equipped with a Falcon 3EC camera (Thermo Fisher Scientific). Optimal particles were observed in grid GD5-3, containing 2 mg/mL S. enterica WbaP. Based on these preliminary results, 4481 movies of SMA30-solubilized S. enterica WbaP were collected on a Titan Krios G3i (Thermo Fisher Scientific, Supplementary file 1B) equipped with a K3 camera (Gatan, Inc). Motion correction, CTF estimation, particle extraction, ab-initio reconstruction, and all refinement steps were conducted using Cryosparc V4 Punjani, 2020 (link); Punjani et al., 2017 (link).

Dodge G.J., Anderson A.J., He Y., Liu W., Viner R, & Imperiali B. (2024). Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle. eLife, 12, RP91125.

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Titan Krios G3i Tomographic Data Collection

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All tomographic data were collected in electron-counting mode using SerialEM software version 3.8 (Mastronarde, 2003 (link)) on a Titan Krios G3i (Thermo Fisher Scientific) equipped with a Gatan energy filter and a K3 electron detector (Gatan). Data were recorded on a Ted Pella waffle pattern grating replica with 2160 lines/mm on a 3mm grid (Ted Pella), at a pixel size of 0.68Å and a target defocus of −1 μm.

Chreifi G., Chen S, & Jensen G.J. (2021). Rapid Tilt-Series Method for Cryo-Electron Tomography: Characterizing Stage Behavior During FISE Acquisition. Journal of structural biology, 213(2), 107716.

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