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Restoration of Membrane Environments for Membrane Proteins for Structural and Functional Studies
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
The cryo-EM samples for the dipole potential measurement were prepared as previously described [28]. Six microliters of the swollen liposome solution were applied to the front side of a glow-discharged holey carbon grid Quantifoil R2/2 (Quantifoil, Germany) and blotted from the back side for 3–6 seconds with a slip of filter paper (Whatman). The specimen was rapidly frozen by plunging into liquid ethane and stored in liquid nitrogen. Images of liposomes within the holes in the carbon film were obtained using a Tecnai F20 electron microscope at 200 keV using 20 or 30 μm objective apertures. The dose for each exposure was about 20 e/Å2. Images were taken at 45,000 or 50,000 magnification and −2.0 to −3.3 µm defocus and recorded on Kodak SO-163 film. This level of defocus was chosen because in simulations a defocus of −2.5 µm gave the largest sensitivity to variations of electron scattering in the membrane interior. Negatives were scanned with a Zeiss SCAI film scanner to an effective pixel size of 2.4 Å. Estimates of the defocus and other parameters of the contrast-transfer function were obtained by fits-to-image power spectra from the carbon surrounding the holes, under the assumption that the amorphous carbon is a random object with constant structure factor magnitudes in the spatial frequency range of 1/30–1/10 Å–1.
Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants
Published in mAbs, 2022
Tatsuhiko Ozawa, Hideki Tani, Yuki Anraku, Shunsuke Kita, Emiko Igarashi, Yumiko Saga, Noriko Inasaki, Hitoshi Kawasuji, Hiroshi Yamada, so-Ichiro Sasaki, Mayu Somekawa, Jiei Sasaki, Yoshihiro Hayakawa, Yoshihiro Yamamoto, Yoshitomo Morinaga, Nobuyuki Kurosawa, Masaharu Isobe, Hideo Fukuhara, Katsumi Maenaka, Takao Hashiguchi, Hiroyuki Kishi, Isao Kitajima, Shigeru Saito, Hideki Niimi
All image processing steps were performed in Relion 3.1.62,63 Micrograph movie frames were aligned and dose-weighted using MotionCor2.64 The contrast transfer function estimation was performed with CTFFIND4,65 and 2073 micrographs were selected. A total of 711242 particles were auto-picked based on the template generated by particle picking using the Laplacian-Gaussian filter and several rounds of two-dimensional (2D) classification. Next, three-dimensional (3D) classification and 2D classification were performed to remove junk particles. One further round of 3D classification was then performed to identify one class containing one spike with three Fabs. This class included 56,247 particles used in the final 3D reconstruction with C3 symmetry to generate a map with a resolution of 3.1 Å after CTF refinement and Bayesian polishing. The reported resolutions are based on the gold-standard Fourier shell correlation curves (FSC = 0.143) criterion. Local resolution was estimated using RELION implemented local resolution estimation. Figures were prepared with Chimera and Chimera X.66
Functional analysis and cryo-electron microscopy of Campylobacter jejuni serine protease HtrA
Published in Gut Microbes, 2020
Urszula Zarzecka, Alessandro Grinzato, Eaazhisai Kandiah, Dominik Cysewski, Paola Berto, Joanna Skorko-Glonek, Giuseppe Zanotti, Steffen Backert
Three µL of HtrACj S197A protein (2 µg/µL) were applied to glow-discharged Quantifoil R2/2 holey carbon grid and vitrified in a Mark IV Vitrobot (FEI). The grids were screened and preliminarily imaged in a Glacios microscope (Thermo Fisher) at 200 keV with a Falcon 2 direct electron camera at 1.2 Å per pixel. 530 movies were collected with 30 frames each and a dose of 1.48 e−/Å2 per frames. After beam induced motion correction with Motioncor261 and Contrast transfer function (CTF) estimation with Gctf,62 527 micrographs were selected for further particle picking and 2D classification performed in RELION-3.63 An initial model generated with EMAN2 was used for 3D classification and refinement.64 This dataset leads to a low resolution 8 Å map (data not shown). In order to improve the global resolution, a second dataset under the same grid condition was acquired with a Titan Krios microscope (Thermo Fisher) at 300 KeV with a K2 direct electron camera at 0.827 Å per pixel in CM01 beamline of ESRF. Here, 1,063 movies were collected with 40 frames each and a dose of 1.2 e−/Å2 per frames. A refinement procedure similar to Glacios data was applied to Krios data set as well. The two datasets were joined following the procedure described65 and the 8 Å map previously obtained was used as initial model for the following step. The resulting 3D refinement and post-processing produced a map with a global resolution of 5.8 Å according to the gold standard FSC with 0.143 cutoff (Table S3 and Fig. S7).
Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein
Published in mAbs, 2021
Curtis D. Hodge, Daniel. J. Rosenberg, Patricia Grob, Mateusz Wilamowski, Andrzej Joachimiak, Greg L. Hura, Michal Hammel
For each specimen, single particle data were picked manually from the micrographs and processed in RELION-3.1.67,68 The final data sets added up to 539, 6548, 693 and 3427 particles from 39, 82, 38 and 59 micrographs for mAb1-2-NPNTD “low” and “high” concentration, and mAb2-4-NPNTD “low” and “high” concentrations, respectively. Contrast transfer function (CTF) was estimated, particle data was extracted as 128 × 128 or 152 × 152 pixel boxes, 2.7 Å/pix, CTF-corrected and normalized before undergoing 2D alignment and classification. Class-average features were interpreted based on the number and arrangement of identifiable antibodies in their densities. Corresponding classes were grouped accordingly and their populations were pooled.