Abstract P-20: Sub 3  Resolution Cryo-EM Structure of Eukaryotic Small (40S) Ribosomal Subunit

Background: The ribosome is a molecular machine that translates mRNAs into proteins. In eukaryotes, ribosome consists of small (40S) and large (60S) subunits. Translation in eukaryotes is a complicated molecular process that involves the formation of various molecular complexes consisting of ribosomal subunits and protein factors. Cryo-EM approaches such as single particle analysis are widely used for structural analysis of components and intermediates of the translation machinery. However, the process of translation in plants is still poorly characterized at a structural level. Here, we present the structure of Triticum aestium small ribosomal subunit obtained at sub 3 resolution that can be used for further structural studies of the translation process in plants. Methods: The structures of the 40S subunits purified from wheat germ extract were obtained using high-resolution single particle cryo-EM. For cryo-EM sample preparation were used Quantifoil R 1.2/1.3 grids coated with an additional 2 nm amorphous carbon film were glow-discharged for 30 seconds at 15 mA using PELCO easiGlow (Ted Pella). 3 µL of the sample were applied onto the grids, blotted for 3 sec at 10°C and 100% humidity, and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher). Cryo-EM data were collected using a Cs-corrected Titan Krios (Thermo Fisher) transmission electron microscope, equipped with a Falcon II direct electron detector. Data were acquired with defocus range of -0.6 to -2.0 at a nominal magnification of 75,000x, giving a calibrated pixel size of 0.86 Å/pixel. The micrographs were recorded as movie stacks. The exposure time for each stack was 1.6 s, corresponding to a total electron dose of ~84 e-/Å2 fractionated into 32 frames (~2.6 e-/Å2 per single frame). A total of 5521 movie stacks was collected. Raw cryo-EM data preprocessing was performed with Warp software (Tegunov et al., 2019). All further data processing steps were performed using the cryoSPARC v3.2.0 software (Punjani et al., 2017). Results: For final cryo-EM map refinement, 140,000 particles were used resulting in 2.7 Å resolution estimated using an FSC=0.143 gold-standard threshold. The obtained structural data clearly demonstrate the peculiarities of the spatial organization of the 40S ribosomal subunit, like the motility of the head relative to the body revealed by 3D variability analysis. Conclusion: The resulting structure was solved at a significantly higher resolution compared to the previously published structure of a plant ribosome (Armache et al., 2010) and will be used as a reference for further studies of translation initiation in plants.

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The Three-dimensional structure of frozen-hydrated nudaurelia capensis β virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127682 ◽

1987 ◽

Vol 45 ◽

pp. 650-651

Author(s):

N. H. Olson ◽

T. S. Baker ◽

Wu Bo Mu ◽

J. E. Johnson ◽

D. A. Hendry

Keyword(s):

South African ◽

Rna Virus ◽

Carbon Film ◽

Three Dimensional ◽

Dimensional Structure ◽

Electron Dose ◽

Total Electron ◽

Three Dimensional Structure ◽

Negative Stain ◽

Dimensional Reconstruction

Nudaurelia capensis β virus (NβV) is an RNA virus of the South African Pine Emperor moth, Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). The NβV capsid is a T = 4 icosahedron that contains 60T = 240 subunits of the coat protein (Mr = 61,000). A three-dimensional reconstruction of the NβV capsid was previously computed from visions embedded in negative stain suspended over holes in a carbon film. We have re-examined the three-dimensional structure of NβV, using cryo-microscopy to examine the native, unstained structure of the virion and to provide a initial phasing model for high-resolution x-ray crystallographic studiesNβV was purified and prepared for cryo-microscopy as described. Micrographs were recorded ∼1 - 2 μm underfocus at a magnification of 49,000X with a total electron dose of about 1800 e-/nm2.

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Three-dimensional reconstruction of the 40S ribosomal subunit from rabbit reticulocytes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128961 ◽

1987 ◽

Vol 45 ◽

pp. 936-937

Author(s):

Neng-Yu Zhang ◽

Terence Wagenknecht ◽

Michael Radermacher ◽

Tom Obrig ◽

Joachim Frank

Keyword(s):

Three Dimensional ◽

Ribosomal Subunit ◽

Uranyl Acetate ◽

Reconstruction Method ◽

Single Exposure ◽

Electron Dose ◽

Ribosomal Subunits ◽

40S Ribosomal Subunit ◽

Dimensional Reconstruction ◽

Rabbit Reticulocytes

We have reconstructed the 40S ribosomal subunit at a resolution of 4 nm using the single-exposure pseudo-conical reconstruction method of Radermacher et al.Small (40S) ribosomal subunits were Isolated from rabbit reticulocytes, applied to grids and negatively stained (0.5% uranyl acetate) in a manner that “sandwiches” the specimen between two layers of carbon. Regions of the grid exhibiting uniform and thick staining were identified and photographed twice (magnification 49,000X). The first micrograph was always taken with the specimen tilted by 50° and the second was of the Identical area untilted (Fig. 1). For each of the micrographs the specimen was subjected to an electron dose of 2000-3000 el/nm2.Three hundred thirty particles appearing in the L view (defined in [4]) were selected from both tilted- and untilted-specimen micrographs. The untilted particles were aligned and their rotational alignment produced the azimuthal angles of the tilted particles in the conical tilt series.

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Subnanometer-resolution structure determination in situ by a hybrid subtomogram averaging – single particle cryoEM – workflow

10.1101/2020.04.24.057299 ◽

2020 ◽

Author(s):

Ricardo Sanchez ◽

Yingyi Zhang ◽

Wenbo Chen ◽

Lea Dietrich ◽

Misha Kudryashev

Keyword(s):

Single Particle ◽

Electron Tomography ◽

Signal To Noise Ratio ◽

Particle Analysis ◽

Electron Dose ◽

Particle Alignment ◽

Zero Degree ◽

Subtomogram Averaging

ABSTRACTCryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.

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The measurement of inner-shell ionization cross aections by electron impact in a TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130961 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1266-1267

Author(s):

Raynald Gauvin ◽

Gilles L'Espérance

Keyword(s):

Electron Impact ◽

Cross Sections ◽

Weight Fraction ◽

Thin Foil ◽

Specimen Thickness ◽

Ionization Cross ◽

Electron Dose ◽

Total Electron ◽

Inner Shell Ionization ◽

Shell Ionization

Values of cross sections for ionization of inner-shell electrons by electron impact are required for electron probe microanalysis, Auger-electron spectroscopy and electron energy-loss spectroscopy. In this work, the results of the measurement of inner-shell ionization cross-sections by electron impact, Q, in a TEM are presented for the K shell.The measurement of QNi has been performed at 120 KeV in a TEM by measuring the net X-ray intensity of the Kα line of Ni, INi, which is related to QNi by the relation :(1)where i is the total electron dose, (Ω/4π)is the fractional solid angle, ω is the fluorescence yield, α is the relative intensity factor, ε is the Si (Li) detector efficiency, A is the atomic weight, ρ is the sample density, No is Avogadro's number, t' is the distance traveled by the electrons in the specimen which is equal to τ sec θ neglecting beam broadening where τ is the specimen thickness and θ is the angle between the electron beam and the normal of the thin foil and CNi is the weight fraction of Ni.

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