Titan krios g4

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The Titan Krios G4 is a high-performance cryo-electron microscope designed for advanced structural biology research. It provides exceptional image quality and resolution, enabling researchers to study the structure and function of proteins and other biological macromolecules at the atomic level.

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15 protocols using titan krios g4

Cryo-EM Structural Analysis of TilA-TslA-TlaA Complex

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Four microliters of purified TilA-TslA-TlaA1-TlaA2 complex at 0.5 mg ml⁻¹ in Buffer B (Supplementary Table 5) was applied onto glow-discharged (60 s, 30 mA) 300 mesh R1.2/1.3 Au grids (Quantifoil). Grids were blotted for 3 s with blot force of +15 at 10 °C and 100% humidity, and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).
Data were collected in counted mode in EER format on a CFEG-equipped Titan Krios G4 (Thermo Fisher Scientific) operating at 300 kV with a Selectris X imaging filter (Thermo Fisher Scientific) with slit width of 10 e^-V and Falcon 4 direct detection camera (Thermo Fisher Scientific) at ×165,000 magnification, with a physical pixel size of 0.693 Å. Movies were recorded at a dose rate of 12.8 e⁻/Å²/s and 3.98 s exposure for a total dose of 50.9 e⁻/Å².

Garrett S.R., Mietrach N., Deme J., Bitzer A., Yang Y., Ulhuq F.R., Kretschmer D., Heilbronner S., Smith T.K., Lea S.M, & Palmer T. (2023). A type VII-secreted lipase toxin with reverse domain arrangement. Nature Communications, 14, 8438.

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Cryo-EM Structural Analysis of SARS-CoV-2 S Protein

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For cryo-EM, 2.25 mg/mL S protein was vitrified on Quantifoil R1.2/1.3 Cu mesh 300 holey carbon grids after a glow discharge of 15 s at 10 mA. For S protein-hACE2 complex (1:1.2 S protein trimer: hACE2 molar ratio) and S protein-mACE2 complex (1:2.1 S protein trimer: mACE2 molar ratio), 2.25 mg/mL mixtures were vitrified on Quantifoil R1.2/1.3 Cu mesh 200 holey carbon grids, coated with 25 nm gold on each side, after a glow discharge of 20 s at 15 mA. All grids were glow discharged using a Pelco easiGlow glow discharge unit (Ted Pella) before 1.8 μL of protein suspension was applied to the surface of the grid at a temperature of 10 °C and a humidity level of >98%. Grids were subsequently blotted (12 s, blot force −10) and plunge frozen into liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific) plunge freezing device. Grids were imaged using a 300 kV Titan Krios G4 transmission electron microscope (TEM) (Thermo Fisher Scientific) equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at 155,000× magnification (calibrated pixel size of 0.5 Å per physical pixel) over a defocus range of −0.5 μm to −2 μm with a total dose of 40 e⁻/Å² using EPU automated acquisition software (ThermoFisher Scientific).

Mannar D., Saville J.W., Poloni C., Zhu X., Bezeruk A., Tidey K., Ahmed S., Tuttle K.S., Vahdatihassani F., Cholak S., Cook L., Steiner T.S, & Subramaniam S. (2024). Altered receptor binding, antibody evasion and retention of T cell recognition by the SARS-CoV-2 XBB.1.5 spike protein. Nature Communications, 15, 1854.

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Cryo-ET Data Acquisition on Titan Krios G4

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Cryo-ET data were collected on a Titan Krios G4 (Thermo Fisher Scientific) system operating at 300 kV equipped with a BioContinuum imaging filter and a K3 direct electron detector (Gatan). Data acquisition was performed using SerialEM^{102 (link),103 (link)}. Owing to the low cell density of the non-concentrated sample, grids were first extensively screened using polygon montages at low magnification (×2,250). After identification of targets, tilt series were acquired using a dose-symmetric tilt scheme^{104 (link)}, covering an angular range of −60° to +60° and a total electron dose of 140–160 e⁻ Å⁻². Tilt series were either acquired at a pixel size of 4.51 Å at the specimen level using 2° angular increments between tilts and a target defocus of −8 µm or at higher magnification (pixel size of 2.68 Å) with 3° angular increments and a defocus ranging from −3 to −6 µm (for sub-tomogram averaging of the ribosome and reconstruction of the cytoskeletal filament). 2D projection images shown in the Article were recorded at a magnification of ×2,250 (pixel size of 39.05 Å) and a target defocus of −200 µm.

Rodrigues-Oliveira T., Wollweber F., Ponce-Toledo R.I., Xu J., Rittmann S.K., Klingl A., Pilhofer M, & Schleper C. (2022). Actin cytoskeleton and complex cell architecture in an Asgard archaeon. Nature, 613(7943), 332-339.

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Cryo-EM of Crosslinked Protein Complexes

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Crosslinked EsaDEG and EsaDEG-EsxBCD samples were diluted to 0.05 mg/ml and 0.2 mg/ml in SEC buffer, respectively. Three microliters of sample were applied onto glowdischarged (60 s, 30 mA) 300-mesh R1.2/1.3 Quantifoil Au grid. Grids were blotted for 2 s in 100 % humidity at 8–12 °C and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Data were collected in counted mode in EER format on a CFEG-equipped Titan Krios G4 (Thermo Fisher Scientific) operating at 300 kV with a Selectris imaging filter (Thermo Fisher Scientific) with slit width of 10 e⁻V and Falcon 4 direct detection camera (Thermo Fisher Scientific) at ×165,000 magnification, with a physical pixel size of 0.723 Å (EsaDEG) or 0.693 Å (EsaDEG-EsxBCD). Movies were collected with a total dose of 57.0 e⁻/Å².

Yang Y., Boardman E., Deme J., Alcock F., Lea S, & Palmer T. (2023). Three small partner proteins facilitate the type VII-dependent secretion export of an antibacterial nuclease. bioRxiv.

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Cryo-iFLM-FIB-milling Workflow for Structural Biology

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After 3D correlative cryo-iFLM-FIB-milling, the grids were imaged using a Titan Krios G4 with a cold Field Emission Gun (E-CFEG)-TEM (Thermo Fisher Scientific, Hillsboro, OR, USA) operated at 300 kV. Images were acquired on a SelectrisX-Falcon 4i direct electron detector (Thermo Fisher Scientific, Hillsboro, OR, USA) in EFTEM mode using a 10-eV slit. Images were captured at magnifications of 125x (1007 Å/pixel) for whole grid mosaic collection, 1950x (63.58 Å/pixel) for whole lamella overview, 6500x (19.26 Å/pixel) for intermediate magnification imaging for feature identification and tracking, and 26000x (4.727 Å/pixel) for data acquisition using SerialEM software (v.4.1). Full frame images of 4096 × 4096 pixels (counting mode at 2~10 e-/px/s (eps) of dose rate over the sample) were collected and saved in the Electron Event Representation (EER) format. Tilt series were collected using a dose symmetric scheme with 3° increments, groups of 2 tilts, a nominal defocus of −8 μm, and a total dose of ~80 e/Å¹. Additional requirements for Montage Parallel Array Cryo-Tomography (MPACT) were 3×3 montage cryo-tilt series with 12 % overlaps in both X and Y, and a total dose of ~50 e/Å¹ per each tile tilt series were collected. The default spiral setting (A_final = 1.5, Period = 3, Turns = 50, Revolutions = 15) was used to spread the dose, as reported previously (16 (link)).

Yang J., Vrbovská V., Franke T., Sibert B., Larson M., Hall A., Rigort A., Mitchels J, & Wright E.R. (2023). Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET. bioRxiv.

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Cryo-ET data acquisition protocol

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Cryo-ET data were acquired on a Thermo Scientific Titan Krios G4 transmission electron microscope operated at 300 kV using SerialEM^{45 (link)}. Raw video frames were recorded on a Thermo Scientific Falcon 4 direct electron detector using the post-column Thermo Scientific Selectris X energy filter. Videos were acquired in Electron Event Representation format^{46 (link)} with a pixel size of 3.03 Å per pixel, an exposure of 3 s and a dose rate of 2.6e⁻ Å⁻² s⁻¹. Tilt series were collected in 3° increments using a dose-symmetric scheme with two tilts per reversal up to 30°, and then bidirectionally to 60°. For a full tilt series, this resulted in an accumulated dose of 104e⁻ Å⁻². Tilt series were acquired between −2.5 and −4.5 µm defocus.

Lacey S.E., Foster H.E, & Pigino G. (2023). The molecular structure of IFT-A and IFT-B in anterograde intraflagellar transport trains. Nature Structural & Molecular Biology, 30(5), 584-593.

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Cryogenic Electron Microscopy Protocol

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Sample volumes of 3.5 µl (8 OD₂₆₀_nm per ml) were applied to grids (Quantifoil, Cu, 300 mesh, R3/3 with 3 nm carbon) which had been freshly glow-discharged using a GloQube (Quorum Technologies) in negative charge mode at 25 mA for 90 s. Sample vitrification was performed using ethane or propane in a Vitrobot Mark IV (Thermo Fisher Scientific), the chamber was set to 4 °C and 100% relative humidity and blotting was done for 3 s with no drain or wait time. Data were collected in an automated manner using EPU v.3.0 on a cold-FEG fringe-free Titan Krios G4 (Thermo Fisher Scientific) transmission electron microscope operating at 300 kV. The camera was operated in electron counting mode and data were collected at a magnification of 96,000× with the nominal pixel size of 0.83 Å and a nominal defocus range of −0.4 to −0.9 μm. A total of 23,349 micrographs in EER format were collected with 5.31 s of exposure (corresponding to a total dose of 50 e per A² on the specimen). No statistical methods were used to predetermine the sample size. The sample size was selected on the basis of a three-day data collection, which was chosen to obtain a sufficient number of particles for data processing.

Dimitrova-Paternoga L., Kasvandik S., Beckert B., Granneman S., Tenson T., Wilson D.N, & Paternoga H. (2024). Structural basis of ribosomal 30S subunit degradation by RNase R. Nature, 626(8001), 1133-1140.

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Cryo-EM Sample Preparation and Data Collection

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QuantiFoil Au (R1.2/1.3 300 mesh, Quantifoil) and UltrAuFoil (R1.2/1.3 300 mesh, Quantifoil) grids underwent glow discharge using easiGlow (PELCO) at 15 mA for 15 s under a pressure of 0.37 mBar. A protein sample of 3 μL was initially placed on the grid’s surface in the Vitrobot Mark IV chamber (maintained at 10 °C with 100% humidity). Subsequently, it was blotted for a duration of 4 s before being rapidly frozen in liquid ethane. Cryogrids were transferred to a 300-kV Titan Krios G4 (Thermo Fisher) for automated data collection by EPU (Thermo Fisher). Movies in EER format were captured using the Falcon IV camera (Thermo Fisher) operating in EC mode, with a nominal magnification set at 75,000× and a pixel size of 1.036 Å. Movies were subjected to a 6-s exposure, accumulating a total electron dose of approximately 40 e⁻/Å². Subsequently, the movie stacks were aligned, dose-weighted, and averaged in cryoSPARC live (36 (link)).

Huang J., Fan X., Jin X., Teng L, & Yan N. (2023). Dual-pocket inhibition of Nav channels by the antiepileptic drug lamotrigine. Proceedings of the National Academy of Sciences of the United States of America, 120(41), e2309773120.

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Cryo-EM Imaging of Purified p97

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Purified p97 was concentrated to approximately 4 mg ml⁻¹ and incubated in the ATP regeneration system (4 mM ribose-5-phosphate, 4 mM MgCl₂, 50 mM KCl, 13.3 U pyruvate kinase, 50 mM phospho-enol pyruvate, 10 mM ATP) for 20 min at 37 °C. Octyl-beta-glucoside at a concentration of 0.05% was added just before plunge-freezing. A 3-μl sample was blotted on glow-discharged Quantifoil Cu R2/1, 200 mesh grids. Plunge-freezing was performed with a Vitrobot Mark IV (Thermo Fisher Scientific) in a chamber equilibrated at 10 °C with 100% humidity. Images were acquired with a Titan Krios G4 (Thermo Fisher Scientific), with a Falcon 4 detector (Thermo Fisher Scientific) mounted after a Selectris energy filter with slit width at 15 eV. A total of 10,011 images were collected using EPU (v 3.1) with aberration-free image shift (AFIS), at ×165,000 magnification (0.72 Å pix⁻¹). Each image had an exposure of 40 e Å⁻², with an exposure rate of 5.41 e pix⁻¹ s⁻¹. The nominal defocus range was from −0.9 to −2.2 μm.

Shein M., Hitzenberger M., Cheng T.C., Rout S.R., Leitl K.D., Sato Y., Zacharias M., Sakata E, & Schütz A.K. (2024). Characterizing ATP processing by the AAA+ protein p97 at the atomic level. Nature Chemistry, 16(3), 363-372.

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SARS-CoV-2 S Trimer Beta Mutant Cryo-EM

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For cryo-EM, SARS-CoV-2 S trimer Beta mutant were deposited on grids at a final concentration of 2 mg/mL. Complexes were prepared by incubating S trimer Beta mutant with VH F6 at a molar ratio of 1:10. Grids were cleaned with H2/O2 gas mixture for 15 s in PELCO easiGlow glow discharge unit (Ted Pella) and 1.8 μL of protein suspension was applied to the surface of the grid. Using a Vitrobot Mark IV (Thermo Fisher Scientific), the sample was applied to either Quantifoil Holey Carbon R1.2/1.3 copper 300 mesh grids or UltrAuFoil Holey Gold 300 mesh grids at a chamber temperature of 10°C with a relative humidity level of 100%, and then vitrified in liquid ethane after blotting for 12 s with a blot force of −10. All cryo-EM grids were screened using a 200-kV Glacios (Thermo Fisher Scientific) TEM equipped with a Falcon4 direct electron detector and data were collection on a 300-kV Titan Krios G4 (Thermo Fisher Scientific) TEM equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at 155,000× magnification (physical pixel size 0.5 Å) over a defocus range of −3 μm to −0.5 μm with a total dose of 40 e – /Å2 using EPU automated acquisition software (Thermo Fisher).

Chen C., Saville J.W., Marti M.M., Schäfer A., Cheng M.H., Mannar D., Zhu X., Berezuk A.M., Banerjee A., Sobolewski M.D., Kim A., Treat B.R., Da Silva Castanha P.M., Enick N., McCormick K.D., Liu X., Adams C., Hines M.G., Sun Z., Chen W., Jacobs J.L., Barratt-Boyes S.M., Mellors J.W., Baric R.S., Bahar I., Dimitrov D.S., Subramaniam S., Martinez D.R, & Li W. (2022). Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains. iScience, 25(8), 104798.

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