Complexes of M4H2K1 Fab and BG505 UFO.664 trimer were purified by SEC to remove unbound Fab and diluted to 0.01 mg/ml in Tris-buffered saline prior to adsorption onto carbon-coated and plasma-cleaned copper mesh grids (Cu400; Electron Microscopy Sciences). Grids were stained with 2% (wt/vol) uranyl formate for ∼60 s and imaged on an FEI Tecnai Spirit microscope operating at 120 keV, equipped with a TVIPS TemCam F416 4k × 4k complementary metal oxide semiconductor (CMOS) camera. Automated data collection was performed using Leginon (123 (link)). Particles were picked using DogPicker in the Appion software suite (124 (link)), extracted using Relion 3.0 (125 (link)), and imported into cryoSPARC v2 (126 (link)). After one round each of 2D and 3D classification, 14,027 particles were included in a 3D refinement with C3 symmetry imposed and a low-pass-filtered volume of ligand-free HIV-1 Env used as the initial model. The final resolution for the negative-stain reconstruction was estimated to be ∼25 Å (Fourier shell correlation cutoff of 0.5).
Temcam f416 4k 4k
Manufactured by TVIPS
Sourced in Germany
The TemCam F416 4k × 4k is a high-resolution direct electron detector designed for transmission electron microscopy (TEM) applications. It features a 4096 × 4096 pixel array and is capable of capturing images at a high frame rate. The device is suitable for a variety of TEM techniques, including low-dose imaging, in-situ experiments, and electron diffraction analysis.
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Lab products found in correlation
3 protocols using temcam f416 4k 4k
1
Negative Stain EM Imaging of M4H2K1 Fab-BG505 UFO.664 Trimer
2
Transmission Electron Microscopy of TMV Complexes
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Samples of TMVCys, TMVCys/Bio, or TMVCys/Bio/[SA]-enzyme complexes (0.2 μg/μl in 10 mM SPP pH 7.0) were adsorbed for 10 min to Formvar®-coated and carbon-sputtered 400 mesh copper grids, washed three times for 1 min on drops of ddH2O and negatively stained using three drops of uranyl acetate (UAc) supplemented with bacitracin (2% (w/v) UAc; 250 mg/ml bacitracin), with 1 min incubation on each drop. TEM (Tecnai G2 Sphera Fei TEM; Fei, Eindhoven, Netherlands) was carried out at 120 kV using a 16 megapixel camera (TemCam-F416 (4k × 4k), TVIPS, Gauting, Germany).
3
TEM Imaging of Hydrogels with TMV
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Hydrogels with and without TMV were embedded in epoxy resin (Spurr) after dehydration of the gels through a solvent series using different acetone : water mixtures (30%, 50%, 75%, 90%). The gels were incubated 20 min at each step. This was followed by staining with 2% uranyl acetate in acetone for one hour and two additional incubations in 100% acetone for 20 min. The samples were then immersed in a solution containing 33% Spurr and 67% acetone overnight without closing the vessels to allow the acetone to evaporate. Before the gels were put into embedding capsules and cured for at least 24 h at 60 °C, the solutions were twice exchanged with 100% Spurr for 1–2 h. The epoxide blocks were trimmed to the gel and ∼80 nm thin sections were prepared using a Leica Ultracut UCT ultramicrotome assembled with a diamond knife (Diatome, Nidau, Switzerland). The sections were placed on Pioloform covered 400 mesh copper grids and investigated using a Tecnai G2 Sphera FEI TEM (FEI, Eindhoven, Netherlands) at 200 kV. TEM images were recorded by a 16 megapixel camera (TemCam-F416 [4k × 4k], TVIPS, Gauting, Germany).
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