For the apo ATM sample, a single dataset was collected at the Memorial Sloan Kettering cryoEM Facility on a Titan Krios microscope operated at 300kEV equipped with a Gatan K3 Summit direct electron detector. Data was acquired using a defocus range of –0.6 to –1.6 µm and a pixel size of 1.056 Å. Each micrograph was acquired using a 3-s exposure and fractionated into 40 frames with a dose of 20 electrons per pixel per second. A total of 9028 micrographs were collected using 3 × 3 image shift. For the ATM-Nc28 sample, a single dataset was collected at Janelia Research Campus on a Titan Krios microscope operated at 300kEV equipped with a Gatan K3 Summit direct electron detector and an energy filter. Data was acquired using a defocus range of –1.0 to –2.5 µm and a pixel size of 1.078 Å. Each micrograph was acquired using a 3-s exposure and fractionated into 40 frames with a dose of 20 electrons per pixel per second. A total of 7,866 micrographs were collected using 3 × 3 image shift.
Titan krios microscope
Manufactured by Ametek
Sourced in Netherlands
The Titan Krios microscope is a high-performance cryo-electron microscope designed for advanced structural biology research. It features a powerful electron beam, high-resolution imaging capabilities, and advanced temperature control systems to enable the detailed study of biological samples in their native state.
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Lab products found in correlation
Vitrobot mark 4, by Thermo Fisher Scientific (11 mentions)
K2 summit, by Ametek (9 mentions)
1.2 1 3 grid, by Quantifoil (4 mentions)
Vitrobot, by Thermo Fisher Scientific (4 mentions)
Fluorinated fos choline 8, by Anatrace (3 mentions)
Gif quantum energy filter, by Ametek (3 mentions)
Laceycarbon films on 300 mesh copper grids, by Agar Scientific (2 mentions)
K2 summit4kx4k detector, by Ametek (2 mentions)
K2 camera, by Ametek (2 mentions)
K2 detector, by Ametek (2 mentions)
K2 summit detector, by Thermo Fisher Scientific (2 mentions)
Epu software, by Thermo Fisher Scientific (2 mentions)
F816 cmos detector, by TVIPS (2 mentions)
K3 camera, by Ametek (2 mentions)
F20 microscope, by Thermo Fisher Scientific (2 mentions)
Au r1 2 1, by Quantifoil (2 mentions)
Quantum energy filter, by Ametek (2 mentions)
Falcon 3 direct electron detector, by Thermo Fisher Scientific (1 mentions)
Tomo software, by Thermo Fisher Scientific (1 mentions)
K2 direct electron detector, by Ametek (1 mentions)
35 protocols using titan krios microscope
1
Cryo-EM Structural Analysis of ATM Kinase
2
Cryo-EM Data Collection for ATM Protein
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For the apo ATM sample, a single 3-day dataset was collected at the Memorial Sloan Kettering cryoEM Facility on a Titan Krios microscope operated at 300kEV equipped with a Gatan K3 Summit direct electron detector. Data was acquired using a defocus range of -0.6 to -1.6µm and a pixel size of 1.056Å. Each micrograph was acquired using a 3 second exposure and fractionated into 40 frames with a dose of 20 electrons per pixel per second. A total of 9,028 micrographs were collected using 3X3 image shift. For the ATM-Nc28 sample, a single 3 day dataset was collected at Janelia Research Campus on a Titan Krios microscope operated at 300kEV equipped with a Gatan K3 Summit direct electron detector and an energy filter. Data was acquired using a defocus range of -1.0 to -2.5µm and a pixel size of 1.078Å. Each micrograph was acquired using a 3 second exposure and fractionated into 40 frames with a dose of 20 electrons per pixel per second. A total of 7,866 micrographs were collected using 3X3 image shift.
3
Cryo-EM Data Collection Protocols
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Single particle cryoEM data were collected on a Titan Krios microscope (‘Titan Krios 1’ at the Astbury Biostructure Laboratory) operating at 300 kV and Falcon III direct electron detector (Thermo Fisher Scientific) operating in integrating mode. Data were collected using EPU software with parameters as in Supplementary Table 1 , based on a published protocol34 (link). Where the sample was tilted during collection, the autofocus and drift measurements were taken in line with the tilt axis.
Tilt series data were collected using a Titan Krios microscope (‘Titan Krios 2’) operating at 300 kV and Bioquantum energy filter (20 eV) K2 direct electron detector (Gatan) operating in counting mode, using Tomo software (Thermo Fisher Scientific). Data were collected with parameters as in Supplementary Table2 .
Tilt series data were collected using a Titan Krios microscope (‘Titan Krios 2’) operating at 300 kV and Bioquantum energy filter (20 eV) K2 direct electron detector (Gatan) operating in counting mode, using Tomo software (Thermo Fisher Scientific). Data were collected with parameters as in Supplementary Table
4
Serotonin-Induced 5-HT3A Receptor Structure
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Functional characterization shows that serotonin-induced 5-HT3AR currents saturate at 30 μM and beyond11 (link),33 (link),34 (link). Therefore, the 5-HT3AR protein (~2.5 mg/mL) was filtered and first incubated with 100 μM serotonin for 30 min. After which, 3 mM Fluorinated Fos-choline 8 (Anatrace) was added and the sample was incubated until blotting35 (link). The sample was blotted twice with 3.5 μL sample each time onto Cu 300 mesh Quantifoil 1.2/1.3 grids (Quantifoil Micro Tools), and immediately after the second blot, the grid was plunge frozen into liquid ethane using a Vitrobot (FEI). The grids were imaged using a 300 kV FEI Titan Krios microscope equipped with a Gatan K2-Summit direct detector camera. Movies containing 40-frames were collected at 130,000x magnification (set on microscope) in super-resolution mode with a physical pixel size of 0.532 Å/pix, dose rate of 3.754 electrons/pix/s, and a total exposure time of 12 seconds. Defocus values of the images ranged from −1.0 to −2.5 µm (input range setting for data collection) as per the automated imaging software Latitute S (Gatan Co.).
5
Cryo-EM Imaging of dArc1 and dArc2 Capsids
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5 μl of dArc1 or dArc2 capsids at a concentration of ~1
mg/ml were applied to glow-discharged continuous carbon lacey grids (Lacey
Carbon Films on 300 Mesh Copper Grids, Agar scientific), blotted and
plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV. Cryo-EM images were
acquired on a 300 keV FEI Titan Krios microscope equipped with a Gatan K2-Summit
4Kx4K detector operated in counting mode. A GIF-quantum energy filter (Gatan)
was used with a slit width of 20 eV to remove inelastically scattered electrons.
Both the dArc1 and dArc2 datasets were collected using SerialEM19 at a nominal magnification of
105,000 with calibrated pixel sizes of 1.211 Å for dArc1 and 1.388
Å for dArc2. Micrographs were recorded as movies divided into 75 frames.
For dArc1 we used a total exposure time of 6.6 s. and an accumulated dose of
35.4 electrons per Å2. For dArc2, the total exposure time used
was 16.8 s and the accumulated dose 34.9 electrons per Å2.
Defocus values ranged from −1.0 to −4.0 μm. Data collection
parameters are summarized inSupplementary Table 1 .
mg/ml were applied to glow-discharged continuous carbon lacey grids (Lacey
Carbon Films on 300 Mesh Copper Grids, Agar scientific), blotted and
plunge-frozen in liquid ethane using a FEI Vitrobot Mark IV. Cryo-EM images were
acquired on a 300 keV FEI Titan Krios microscope equipped with a Gatan K2-Summit
4Kx4K detector operated in counting mode. A GIF-quantum energy filter (Gatan)
was used with a slit width of 20 eV to remove inelastically scattered electrons.
Both the dArc1 and dArc2 datasets were collected using SerialEM19 at a nominal magnification of
105,000 with calibrated pixel sizes of 1.211 Å for dArc1 and 1.388
Å for dArc2. Micrographs were recorded as movies divided into 75 frames.
For dArc1 we used a total exposure time of 6.6 s. and an accumulated dose of
35.4 electrons per Å2. For dArc2, the total exposure time used
was 16.8 s and the accumulated dose 34.9 electrons per Å2.
Defocus values ranged from −1.0 to −4.0 μm. Data collection
parameters are summarized in
6
Cryo-ET Imaging of Protein Particles
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Cryo-ET tilt series were acquired using SerialEM software53 (link) from +60° to −60° with a step of 3°, on an FEI Titan Krios microscope (300 kV) equipped with a Gatan K2 camera. At each tilt angle, we collected movies containing 8 frames, with a sum dose of ~3 e− Å−2 s−1, and the total dose for every tilt series (+60° to −60°) was 120 e− Å−2. The pixel size was 1.25 Å. The movies were first motion-corrected by MotionCor247 (link) and then imported into Etomo54 (link) for alignment and reconstruction. The position of protein particles inside the ice was manually identified and plotted13 (link).
7
Cryo-EM Analysis of Kq-KlebC Complex
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KqTolC-KlebC51-245 complex was applied to Quantifoil 300 mesh Cu, 1.2/1.3, grids at concentrations of 0.5 mg ml−1 and 0.75 mg ml−1 for native and formaldehyde cross-linked samples, respectively. Samples (3 μl) were vitrified using a vitrobot Mark IV (Thermo Fisher) with a blot time of 3 s, blot force of −2, and wait time of 30 s. Cryo-EM data were acquired on a 300-kV Titan Krios microscope, equipped with a K2 detector (Gatan), recording in counting mode. A pixel size of 0.822 Å was used to collect videos of 30 frames with a dose rate of 4.8 and 3.7 electrons/Å2/s, from an 8-s exposure, resulting in a total dose of 38 and 29 electrons/Å2 for native and formaldehyde cross-linked samples, respectively. For further collection parameters, see Supplementary Table 2 .
8
Cryo-EM Grid Preparation and Data Acquisition
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For grid preparation, complex samples were used either at a concentration of 1.2 mg/ml or 1.6 mg/ml with the same exchanged buffer as described above. The sample (3.5 μl) was applied to glow-discharged holey carbon grid (CryoMatrix Amorphous alloy film R1.2/1.3, 300 mesh) and subsequently vitrified using an FEI Vitrobot Mark IV at 4 °C and 100% humidity. The grids were blotted for 2.5 s at a force of −1 and vitrified by plunge freezing into liquid ethane cooled by liquid nitrogen at −180 °C.
Cryo-EM data were collected on an FEI Titan Krios microscope using a K2 camera positioned post a Gatan GIF quantum energy filter, with a slit width of 20 eV. Micrographs were recorded in super-resolution mode at a magnified physical pixel size of 0.52 Å, with defocus values ranging from −1.0 to −2.0 μm. The total exposure time was 8.0 s and intermediate frames were recorded in an accumulated dose of 60 electrons per Å2 and a total of 40 frames per micrograph. Data acquisition was done using SerialEM64 (link).
Cryo-EM data were collected on an FEI Titan Krios microscope using a K2 camera positioned post a Gatan GIF quantum energy filter, with a slit width of 20 eV. Micrographs were recorded in super-resolution mode at a magnified physical pixel size of 0.52 Å, with defocus values ranging from −1.0 to −2.0 μm. The total exposure time was 8.0 s and intermediate frames were recorded in an accumulated dose of 60 electrons per Å2 and a total of 40 frames per micrograph. Data acquisition was done using SerialEM64 (link).
9
Cryo-EM Structural Determination of CPSF-PAS RNA Complex
10
Cryo-EM Imaging of GspD Protein Complex
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All tilt series were collected using 5° angular step, from −50° to +50°, defocus from −1.5 to −5 µm, and a total dose of about 100 e−/Å2. For the GspDα on the inner and outer membrane dataset, the sample was imaged using the bidirectional data collection scheme (starting angle −30°) on a 300 kV FEI Titan Krios microscope with a Gatan K2 Summit direct electron detector camera, using the SerialEM software. The magnification was 81,000×, with a pixel size of 1.76 Å. For the other datasets and the control dataset, the sample was imaged using a dose symmetric data collection scheme on a 200 kV FEI Glacios microscope with a Falcon 4 direct electron detector camera, using the Tomography software. The magnification was 92,000×, with a calibrated pixel size of 1.52 Å (Supplementary Table 1 ).
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