Fei titan krios microscope

Manufactured by Thermo Fisher Scientific

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The FEI Titan Krios microscope is a high-performance cryo-electron microscope designed for advanced structural biology research. It features a powerful electron beam, sophisticated optics, and a cryogenic sample stage to enable the high-resolution imaging of biological macromolecules and complexes.

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Titan Krios (617 citations) Titan Krios microscope (327 citations) Krios microscope (40 citations) FEI Titan Krios (34 citations) FEI Titan Krios electron microscope (17 citations) FEI Titan Krios transmission electron microscope (5 citations) FEI Krios microscope (2 citations) FEI Titan microscope (2 citations)

25 protocols using fei titan krios microscope

Structural Determination of gH/gL-1D8-AMMO1 Complex

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We reconstituted the complex of the gH/gL bound to the 1D8 Fab and the AMMO1 Fab at 1:1:1 molar ratio. Aliquots of complexes (4 μl, 0.5 mg/ml, in buffer containing 20 mM Tris pH 8.0 and 150 mM NaCl) were applied to glow-discharged holey carbon grids (Quantifoil grid, Au 300 mesh, R1.2/1.3). The grids were then blotted and plunge-frozen into liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific). Images for complexes were recorded using 300 kV FEI Titan Krios microscope (Thermo Fisher Scientific) at Tsinghua University. We collect ~5000 movies for complexes at a nominal magnification of 29,000× and at a defocus range between −1.2 and −1.5 μm. Each movie has a total accumulate exposure of 50 e − /Å² fractionated in 32 frames of 175 ms exposure. The final image was binned 2-fold to a pixel size of 0.97 Å. Motion Correction (MotionCor2 v.1.2.6)^{66 (link)}, CTFestimation (GCTF v.1.18)^{67 (link)} and non-templated particle picking (Gautomatch v.0.56, http://www.mrc-lmb.cam.ac.uk/kzhang/) were automatically executed by TsingTitan.py program. Sequential data processing and 2D projection of gH/gL-1D8-AMMO1 model was carried out on RELION3.1^{68 (link)}.

Zhu Q.Y., Shan S., Yu J., Peng S.Y., Sun C., Zuo Y., Zhong L.Y., Yan S.M., Zhang X., Yang Z., Peng Y.J., Shi X., Cao S.M., Wang X., Zeng M.S, & Zhang L. (2021). A potent and protective human neutralizing antibody targeting a novel vulnerable site of Epstein-Barr virus. Nature Communications, 12, 6624.

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Cryo-EM Sample Preparation for Protein Complex

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Aliquots of NC22/17-HA 131E ectodomain (4 uL, 0.18 mg/mL, in 20 mM Tris pH 8.0, 150 mM NaCl.) were applied to graphene oxide (GO) grids (GO on Quantifoils R1.2/1.3 400 mesh copper grids, R1.2/1.3). In the Vitrobot Mark IV (Thermo Fisher Scientific), the humidity was set at 100%, the protein solution was applied to the grid and there was a wait of 1 s before blotting. A blot force of 0 and blot time of 2 s were applied to blot the grid after waiting. After blotting, the grid was plunged into precooled liquid ethane at a liquid nitrogen temperature (69 (link)).
Images for complex were recorded using FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc.) at Shanxi Academy of Advanced Research and Innovation. The automated software (EPU)3 was used to collect 5,746 movies for complex in super-resolution mode at a nominal magnification of 105,000× with a calibrated pixel size of 0.85 Å and at a defocus range between −1.0 and −2.0 um. Each movie has a total accumulate exposure of 60 e-/Å2 fractionated in 32 frames of 1.38 s exposure (70 (link)).

Sun H., Deng G., Sun H., Song J., Zhang W., Li H., Wei X., Li F., Zhang X., Liu J., Pu J., Sun Y., Tong Q., Bi Y., Xie Y., Qi J., Chang K.C., Gao G.F, & Liu J. (2022). N-linked glycosylation enhances hemagglutinin stability in avian H5N6 influenza virus to promote adaptation in mammals. PNAS Nexus, 1(3), pgac085.

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Cryo-EM Imaging of Molecular Complexes

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Aliquots of 3 μL of complex were applied to glow-discharged Quantifoil^® (1.2/1.3) grids. The grids were blotted for 3 seconds at 100% humidity with an offset of 3 and plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Grids were imaged on a 300 kV FEI Titan Krios microscope (Thermo Fisher) located at the Stanford cEMc facility and equipped with a K3 camera and energy filter (Gatan). Movies were collected at a calibrated magnification of ×130,000, corresponding to 0.653 Å per physical pixel. The dose was set to a total of 50.6 electrons per Å². Automated data collection was carried out using SerialEM^{24 (link)} with a nominal defocus range set from −0.8 to −3.0 μM. 21,154 movies were collected to compensate for low particle density on the imaged grids.

, & Moulton D. (2000). A stitch in time. CMAJ: Canadian Medical Association Journal, 163(2), 198.

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Cryo-EM of DARP14-3G124Mut5-sfGFP

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Fresh fractions from the Superose 6 Increase column containing DARP14-3G124Mut5 bound with sfGFP V206A were pooled and concentrated to 2 mg/mL. The sample was diluted to 1 mg/mL and final buffer composition of 10 mM Tris pH 7.5, 500 mM NaCl, 1 mM DTT, 0.5% glycerol immediately prior to freezing. Quantifoil 200 mesh 1.2/1.3 copper grids (Electron Microscopy Sciences) was treated with 0.1% poly-lysine (Sigma-Aldrich) for 4–6 h prior to freezing and cleaned of excess poly-lysine by washing with filtered water three times. 2.5 μL of sample was applied to the grids without glow discharging and frozen using a Vitrobot Mark IV (FEI). 1,929 movies were collected on a FEI titan Krios microscope (Thermo Fisher) with a Gatan K2 Summit direct electron detector in counting mode with image shift at a pixel size of 1.07 Å, and defocus values around −2.5 μm. Movies with 40 frames were collected over 8 s with ~7.00 e⁻* A⁻²* s⁻¹ dose rate.

Liu Y., Huynh D.T, & Yeates T.O. (2019). A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. Nature Communications, 10, 1864.

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Cryo-EM structural analysis of SARS-CoV-2 Spike

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Aliquots of complexes (4 μl, in buffer containing 20 mM Tris, pH 8.0, and 150 mM NaCl) of the SARS-CoV-2 spike ectodomains (WT 1.65 mg/ml, BA.1 2 mg/ml, BA.4 1.3 mg/ml, respectively) and Fab (6-2C Fab, 10-5B Fab) were applied to glow-discharged holey carbon grids (Quantifoil grid, Cu 300 mesh, R1.2/1.3). The Fab fragments were mixed with the SARS-CoV-2 spike trimer at a molar ratio of 1.2:1. The grids were then blotted for 2 s and immediately plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The cryo-EM data of the complexes were collected with an FEI Titan Krios microscope (Thermo Fisher Scientific) at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc., Pleasanton, CA, USA) at Tsinghua University. A total of 5494 movies were collected with SerialEM v 4.0.4, with a magnification of 29000 and defocus range between −1.3 and −1.5 μm. Each movie had a total accumulated exposure of 50 e-/Å² fractionated in 32 frames of 2.13 s exposures. The stacks were binned twofold, resulting in a pixel size of 0.97 Å/pixel.

Liu Y., Wang Z., Zhuang X., Zhang S., Chen Z., Zou Y., Sheng J., Li T., Tai W., Yu J., Wang Y., Zhang Z., Chen Y., Tong L., Yu X., Wu L., Chen D., Zhang R., Jin N., Shen W., Zhao J., Tian M., Wang X, & Cheng G. (2023). Inactivated vaccine-elicited potent antibodies can broadly neutralize SARS-CoV-2 circulating variants. Nature Communications, 14, 2179.

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Cryo-EM Imaging of BG505-F14 SOSIP

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Cryo-EM imaging was performed on a FEI Titan Krios microscope (Thermo Fisher Scientific) operated at 300 kV. Data collection images were acquired with a Falcon 3EC Direct Electron Detector operated in counting mode with a calibrated physical pixel size of 1.08 Å with a defocus range between −1.0 and −3.5 µm using the EPU software (Thermo Fisher Scientific). No energy filter or C_s corrector was installed on the microscope. The dose rate used was ∼0.8 e⁻/Å²·s to ensure operation in the linear range of the detector. The total exposure time was 60 s, and intermediate frames were recorded every 2 s giving an accumulated dose of ∼42 e⁻/Å² and a total of 30 frames per image. A total of 2350 images for BG505-F14-SOSIP and 2060 images for BG505-F14/Vt8-SOSIP were collected over 2 days, respectively.

Henderson R., Lu M., Zhou Y., Mu Z., Parks R., Han Q., Hsu A.L., Carter E., Blanchard S.C., Edwards R.J., Wiehe K., Saunders K.O., Borgnia M.J., Bartesaghi A., Mothes W., Haynes B.F., Acharya P, & Munir Alam S. (2020). Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements. Nature Communications, 11, 520.

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Cryo-EM Imaging of Molecular Complexes

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Caveney N.A., Saxton R.A., Waghray D., Glassman C.R., Tsutsumi N., Hubbard S.R, & Christopher Garcia K. (2023). Structural basis of Janus kinase trans-activation. Cell reports, 42(3), 112201.

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Cryo-EM of Purified Protein Complexes

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Purified complex (3.5 μl, 1.9 μΜ) was applied to glow-discharged UltraAuFoil 300 mesh R1.2/1.3 grids (Electron Microscopy Science), incubated for 15 s at 23 °C and 100% humidity, blotted with a blot force of 0 for 12 s then plunge-vitrified into liquid ethane using a FEI Vitrobot Mark IV (Thermo Fisher). A total of 6,429 super-resolution videos were collected at a nominal magnification of ×105,000 on a FEI Titan Krios microscope (Thermo Fisher), equipped with a K3 direct electron detector and BioQuantum energy filter (Gatan) and set to a slit width of 20 eV. Collection was performed semiautomatically using SerialEM at a dose rate of 8.0 e^– pixel⁻¹ s⁻¹ for a total dose of 68 e^– Å^–2 over 118 frames^{61 (link)}. Dose-fractionated image stacks were motion corrected, dose weighted and 2× binned to the physical pixel size of 0.835 Å by MotionCor2 in the package SCIPION^{62 (link),63 (link)}. A defocus range of −0.8 to −2.0 μm was applied.

Li Y.L., Langley C.A., Azumaya C.M., Echeverria I., Chesarino N.M., Emerman M., Cheng Y, & Gross J.D. (2023). The structural basis for HIV-1 Vif antagonism of human APOBEC3G. Nature, 615(7953), 728-733.

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Structural Analysis of SARS-CoV-2 Spike-Fab Complex

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Aliquots of complex of SARS-CoV-2 spike ectodomains and P36-5D2 Fab (4 μl, 1.6 mg/ml, in buffer containing 20 mM Tris, pH 8.0, and 150 mM NaCl) were applied to glow-discharged holey carbon grids (Quantifoil grid, Au 300 mesh, R1.2/1.3). The grids were then blotted for 2 s and plunge-frozen into liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific).
Images for complex were recorded using the FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc., Pleasanton, CA, USA) at Tsinghua University. The automated software AutoEMation2 (53 (link)) was used to collect 4,534 movies for complex of SARS-CoV-2 spike ectodomains and P36-5D2 Fab in super-resolution mode at a nominal magnification of ×29,000 and at a defocus range between −1.5 and −1.8 μm. Each movie has a total accumulated exposure of 50 e-/Å² fractionated in 32 frames of 2.13-s exposures. The final image was binned twofold to a pixel size of 0.97 Å. The data collection statistics are summarized in Supplementary Table S3.

Shan S., Mok C.K., Zhang S., Lan J., Li J., Yang Z., Wang R., Cheng L., Fang M., Aw Z.Q., Yu J., Zhang Q., Shi X., Zhang T., Zhang Z., Wang J., Wang X., Chu J.J, & Zhang L. (2021). A Potent and Protective Human Neutralizing Antibody Against SARS-CoV-2 Variants. Frontiers in Immunology, 12, 766821.

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Omicron BA.1 Spike-Nanobody Complex

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The trimeric spike of Omicron BA.1 at concentration of 1.5 mg/mL was incubated with 3-fold molar excess of 3-2A2-4 at room temperature for 1 hours. 4 μL of mixture was applied to glow-discharged holey carbon grids (Quantifoil grid, Cu 300 mesh, R1.2/1.3). The Quantifoil grids were blotted for 2 seconds with filter paper in 100% relative humidity at 8 °C and plunged into the liquid ethane to freeze samples using FEI Vitrobot system (FEI). Images for nanobody-spike trimer complexes were recorded using FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc.) at Tsinghua University. The automated software (AutoEMation2, PMID: 15797731) was used to collect 4795 movies for nanobody-spike trimer complexes in super-resolution mode at a nominal magnification of 81,000× with a pixel size of 1.0979 Å at a defocus range between -1.5 and-1.8 μm. Each movie has a total accumulate exposure of 50 e-/Å2 fractionated in 32 frames of 175 ms exposure.

Li M., Ren Y., Aw Z.Q., Chen B., Yang Z., Lei Y., Cheng L., Liang Q., Hong J., Yang Y., Chen J., Wong Y.H., Wei J., Shan S., Zhang S., Ge J., Wang R., Dong J.Z., Chen Y., Shi X., Zhang Q., Zhang Z., Chu J.J., Wang X, & Zhang L. (2022). Broadly neutralizing and protective nanobodies against SARS-CoV-2 Omicron subvariants BA.1, BA.2, and BA.4/5 and diverse sarbecoviruses. Nature Communications, 13, 7957.

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