For the reconstitution of TnsC filaments bound to TniQ, wild-type TnsC protein was diluted to a final concentration of 15 μM in a buffer containing 20 mM HEPES-KOH pH 7.5, 100 mM KCl, 10 mM MgCl2, 1 mM DTT and 1 mM ATP and mixed at a ratio of 1:25 (TnsC:DNA) with a double-stranded 69-bp duplex DNA oligonucleotide (Table S3 ) and at a 1:2 ratio (TnsC:TniQ) with TniQ in a 22.4 μL reaction volume. For preparation of cryo-EM grids, 3.0 μL of sample was applied to glow-discharged 200-mesh copper 2 nm C R1.2/1.3 cryo-EM grids (Quantifoil Micro Tools), blotted 1 s at 100% humidity, 4°C, plunge frozen in liquid ethane (using a Vitrobot Mark IV plunger, FEI) and stored in liquid nitrogen. Cryo-EM data collection was performed on a FEI Titan Krios G3i microscope (University of Zurich, Switzerland) operated at 300 kV equipped with a Gatan K3 direct electron detector in super-resolution counting mode. A total of 10,436 movies were recorded at a calibrated magnification of 130,000× resulting in super-resolution pixel size of 0.325 Å. Each movie comprised 36 subframes with a total dose of 66.036 e− Å−2. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher Scientific) with three shots per hole at −1.0 μm to −2.4 μm defocus (0.2 μm steps).
Titan krios g3i microscope
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
The Titan Krios G3i microscope is a high-performance cryo-electron microscope (cryo-EM) designed for advanced structural biology research. It is equipped with a stable and powerful electron beam, advanced optics, and a state-of-the-art detection system. The Titan Krios G3i enables researchers to obtain high-resolution, three-dimensional images of biological macromolecules and complexes, providing insights into their structure and function.
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Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (20 mentions)
K3 direct electron detector, by Ametek (7 mentions)
K3 bioquantum direct electron detector, by Ametek (6 mentions)
Epu software, by Thermo Fisher Scientific (4 mentions)
K3 camera, by Ametek (3 mentions)
Vitrobot mark 4 plunger, by Thermo Fisher Scientific (2 mentions)
Epu automated data acquisition software for single particle analysis, by Thermo Fisher Scientific (2 mentions)
Fei epu, by Thermo Fisher Scientific (2 mentions)
K3 bioquantum camera, by Ametek (2 mentions)
Holey carbon grid, by Quantifoil (2 mentions)
K3 direct detector, by Ametek (2 mentions)
Vitrobot mark 4 system, by Thermo Fisher Scientific (2 mentions)
Bioquantum energy filter, by Ametek (2 mentions)
Au r1 2 1, by Quantifoil (1 mentions)
K2 summit, by Thermo Fisher Scientific (1 mentions)
Graphene oxide, by Merck Group (1 mentions)
Polylysine, by Merck Group (1 mentions)
Vitrobot 4, by Thermo Fisher Scientific (1 mentions)
Solarus plasma cleaner, by Ametek (1 mentions)
Talos l120c, by Thermo Fisher Scientific (1 mentions)
39 protocols using titan krios g3i microscope
1
Cryo-EM Reconstitution of TnsC-TniQ Filaments
2
Cryo-EM Analysis of NSP12-7-8 Complex
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For subsequent EM analyses, NSP12‐7‐8 complex (0.8 mg mL−1) was incubated with GOS at a 1:10 molar ratio at 4 °C for 1 h. A 4 µL aliquot of GOS‐bound complex was applied to a glow‐discharged 300 mesh grid (Quantifoil Au R1.2/1.3) supported with a thin layer of reduced graphene oxide, blotted with filter paper for 3.0 s and plunge‐frozen in liquid ethane using an FEI Vitrobot Mark IV. Cryo‐EM micrographs were collected on a 300 kV Titan Krios (G3i) microscope (FEI) equipped with a K3 direct detection camera and a Gatan image filter (GIF: slit width of 20 eV). The micrographs were collected at a calibrated magnification of ×130 000, yielding a pixel size of 0.27 Å in super‐resolution mode. In total, 10 683 movies were collected at an accumulated electron dose of 50e−Å−2 s−1 on each micrograph, which was fractionated into a stack of 32 frames with a defocus range of −1.0 to −2.0 µm.
3
Structural Characterization of Glutamine Synthetases
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Purified protein samples of CsGSIb (4 μL, 0.02 mg/mL) and GmGSβ2 (4 μL, 0.02 mg/mL) in 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 1.5 mM β-mercaptoethanol were negatively stained with uranyl acetate 1 % (w/v) on carbon-film 400 mesh copper grids. Samples were imaged using a FEI T12 operated at 120 keV with a 3.236 Å pixel size, 68,000× nominal magnification, and defocus range about 1.5 μm. For cryo-EM, 3 μL of CsGSIb (0.1 mg/mL and 0.5 mg/mL) and GmGSβ2 (0.1 mg/mL) were added onto glow-discharged Quantifoil R1.2/1.3 100 holey-carbon Cu grids with a Vitrobot Mark IV (Thermo Fisher Scientific). The grids were blotted for 3.5 s at 8 °C with 100 % humidity, and then plunged frozen into liquid ethane cooled by liquid nitrogen. Cryo-grids were first screened on a FEI TF20 operated at 200 keV. Images of CsGSIb and GmGSβ2 were collected using Titan Krios G3i microscope (FEI) operated at 300 kV with a Gatan K2 Summit direct detection camera. Two datasets were acquired using the SerialEM in super-resolution mode with a nominal magnification of 29,000 x, yielding a pixel sizes of 0.505 Å with a total dose of 51 e/ Å2. The defocus ranges were set from −1.6 μm to −2.3 μm.
4
Cryo-EM Structure of BabSPARTA-gRNA Complex
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Purified BabSPARTA was mixed with a 5′-phosphorylated RNA guide (oBK084, 5′-UGACGGCUCUAAUCUAUUAGU-3′) in assembly buffer (20 mM HEPES pH 7.5, 125 mM KCl, 2 mM MgCl2). The final sample contained 20 μM BabSPARTA and 30 μM gRNA (1:1.5 molar ratio) in a total volume of 80 μl. The volume was incubated at 50°C for 1 hour, centrifuged at 18000 rpm for 10′ at room temperature and directly used for cryo-EM grid preparation.
3.5 μl of sample was applied to a freshly glow discharged 200-mesh Au R1.2/1.3 grid (Quantifoil Micro Tools), incubated for 5 s, blotted for 6 s at 100% humidity, 4 °C, plunge frozen in liquid ethane (using a Vitrobot Mark IV plunger, FEI) and stored in liquid nitrogen. cryo-EM data collection was performed on a FEI Titan Krios G3i microscope (University of Zurich, Switzerland) operated at 300 kV and equipped with a Gatan K3 direct electron detector in super-resolution counting mode. A total of 14 332 movies were recorded at 130 000× magnification, resulting in a super-resolution pixel size of 0.325 Å. Each movie comprised 36 subframes with a total dose of 58.005 e−/Å2. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher Scientific) with three shots per hole at -1.0 mm to -2.4 mm defocus (0.2 mm steps).
3.5 μl of sample was applied to a freshly glow discharged 200-mesh Au R1.2/1.3 grid (Quantifoil Micro Tools), incubated for 5 s, blotted for 6 s at 100% humidity, 4 °C, plunge frozen in liquid ethane (using a Vitrobot Mark IV plunger, FEI) and stored in liquid nitrogen. cryo-EM data collection was performed on a FEI Titan Krios G3i microscope (University of Zurich, Switzerland) operated at 300 kV and equipped with a Gatan K3 direct electron detector in super-resolution counting mode. A total of 14 332 movies were recorded at 130 000× magnification, resulting in a super-resolution pixel size of 0.325 Å. Each movie comprised 36 subframes with a total dose of 58.005 e−/Å2. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (ThermoFisher Scientific) with three shots per hole at -1.0 mm to -2.4 mm defocus (0.2 mm steps).
5
Cryo-EM Structural Analysis
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Data collection was performed with the Titan Krios G3i microscope (Thermo Fisher Scientific, USA) equipped with a K3 BioQuantum direct electron detector (Gatan, USA). Movies were collected via FEI EPU (Thermo Fisher Scientific, USA) automated data collection software at a total dose of ~50 e−/Å2 fractionated over 40 frames with defocus values ranging from −1.5 to −2.5 μm. A super-resolution mode was used with the final pixel size at 0.53 Å.
6
Cryo-ET Data Collection with Titan Krios
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Movies of cryo-ET data were collected using the same Titan Krios G3i microscope (Thermo Fisher Scientific, USA) equipped with a K3 BioQuantum direct electron detector (Gatan, USA). The cryo-EM movies were automatically collected using Thermo Fisher Tomography (Thermo Fisher Scientific, USA) with a slit width of 20 eV on the energy filter. The tilt series were acquired using a bidirectional acquisition scheme from −60° to +60° with a 3° increment (59 (link)). The total dose was 158 e−/Å2, the final pixel size was 1.34 Å/pix, and the target defocus was set to −4.0 μm.
7
NSD2-Nucleosome Complex Structural Analysis
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The reconstituted NSD2-nucleosome complex was applied to a freshly glow-discharged Quantifoil holey carbon grid (R1.2/1.3, 300 mesh, Cu/Rh grid for the sample with 25 mM KCl, and Au grid for the sample with 50 mM KCl), subjected to blotting for 4 s at 4 °C in 100% humidity, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Grid images were obtained using a 300 kV Titan Krios G3i microscope (Thermo Fisher Scientific) equipped with a K3 direct electron detector (Gatan) installed at the University of Tokyo, Japan. Data sets were acquired with SerialEM software, with a defocus range of −0.8 to −1.6 μm. Data acquisition statistics are shown in Supplementary Table 1 .
8
Cryo-EM Analysis of Prion Protein Fibrils
9
Cryo-EM structure determination of bacteriophage terminase
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Quantifoil R2/1 holey carbon copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) were plasma-cleaned for 40 s in Solarus plasma cleaner (Gatan, Inc.) using H2O2 plasma. A total of 2.5 μl of purified terminase at 0.5 mg/ml was applied to the grid prior to freezing using a Vitrobot IV (Thermo Fisher Scientific) automated plunge-freezing robot. For the ATPγS stalled sample, ATPγS was added to get a 1 mM final concentration prior to freezing. Grids were loaded into a Titan Krios G3i microscope (Thermo Fisher Scientific) housed at SCSB center at UTMB, and 500 micrographs for each sample were collected at 300 keV on a Falcon III direct electron detector operating in linear mode at 75K nominal magnification at the detector, thus corresponding to a pixel size of 1.1 Å/pixel. The defocus range was set from −1.4 to −3.2 μm. Each area was exposed to a total dose of 50 electrons/Å2 in 0.97 s. 38 fractions were recorded per exposure. Collected movie fractions were processed in CryoSPARC v4.2 (59 (link)). Both datasets contained ∼244 particles per micrograph on average. 18 925 particles of apo terminase and 24 408 particles of ATPγS stalled terminase went into well-aligned 2D classes after several rounds of classification.
10
Cryo-EM Data Collection Optimization
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Cryo-EM grids were examined in a low-dose mode on a Talos L120C transmission electron microscope (TEM; Thermo Fisher Scientific) for screening. Data collection on high-quality grids in all conditions was performed with the same Titan Krios G3i microscope (Thermo Fisher Scientific) equipped with a K3 BioQuantum direct electron detector (Gatan). Special care was taken to perform a coma-free alignment on the microscope.
Movies were collected via FEI EPU (Thermo Fisher Scientific) (56 (link)) automated data collection software at a total dose of 60 e−/Å2 fractionated over 50 frames in a defocus range of −1.0 to −1.5 µm. A superresolution mode was used with a final pixel size at 0.53 Å. The numbers of the collected movies under different conditions are listed inSI Appendix, Fig. S2B and Table S1 .
Movies were collected via FEI EPU (Thermo Fisher Scientific) (56 (link)) automated data collection software at a total dose of 60 e−/Å2 fractionated over 50 frames in a defocus range of −1.0 to −1.5 µm. A superresolution mode was used with a final pixel size at 0.53 Å. The numbers of the collected movies under different conditions are listed in
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