A posteriori correction of camera characteristics from large image data sets

Afanasyev, Pavel; Ravelli, Raimond B. G.; Matadeen, Rishi; Carlo, Sacha De; Duinen, G. van; Alewijnse, Bart; Peters, Peter J.; Abrahams, Jan Pieter; Portugal, Rodrigo Villares; Schatz, Michael; Heel, Marin van

doi:10.1038/srep10317

Cited by 26 publications

(22 citation statements)

References 23 publications

(41 reference statements)

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“…Gain and dark references were calculated using the method described by Afanasyev et al (Afanasyev et al, 2015) and used to correct the collected movies in cisTEM (Grant et al, 2018). All further image processing was done using cisTEM.…”

Section: Cryo-em and Image Processingmentioning

confidence: 99%

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Bao

Loerch

Ling

et al. 2020

Preprint

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Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

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Section: Cryo-em and Image Processingmentioning

confidence: 99%

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Bao

Loerch

Ling

et al. 2020

Preprint

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“…We applied a flat-fielding technique (adapted from Ref. (Afanasyev et al 2015)) to compensate for spatial inhomogeneities in the CCG, caused by variations in the macromolecular density across the FOV, which, as searches for a decoy target Searching 547 images of the cellular periphery with the ribosomal large subunit (LSU) as the target, we found a total of 9,359 peaks, many extending well above the noise floor ( Fig. 2A).…”

mentioning

confidence: 99%

Label-free single-instance protein detection in vitrified cells

Choi

Lippincott‐Schwartz

et al. 2020

Preprint

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A general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of imagescalculated on the basis of known structural data-as search templates to detect targets as small as the "head" domain (350 kDa) of the ribosome's small subunit in single-tilt electron cryomicrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryoelectron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling. Main:Finding molecular components in intact cells has driven the development of biological imaging techniques, some of which use specific labels, such as fluorescent antibodies, while others rely on a target's intrinsic properties such as its shape (Oliver 1973) and structure (Rickgauer, Grigorieff, and Denk 2017). Labelling is frequently incomplete or nonspecific, can alter the target's interactions with other molecules, provides only moderate precision of localization and

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“…An a posteriori camera correction is then applied to minimize spurious correlations owing to camera imperfections (Afanasyev et al, 2015). Large data sets collected with the same camera reflect the characteristics of each individual pixel.…”

Section: Camera Correction and Data Pre-processingmentioning

confidence: 99%

“…This makes it necessary to apply extreme low-pass filters prior to movie alignments. An a posteriori camera correction (x2.3) significantly minimizes spurious correlations from such fixed background patterns and thus improves movie alignments (Afanasyev et al, 2015). We perform translational movie alignments on the full movie frames or, as was performed here, on extracted patch movies (1280 Â 1280).…”

Section: Movie Alignmentsmentioning

confidence: 99%

“…The collection of large data sets is essential to obtain highresolution results by bringing even subtle differences between the images to statistical significance. For example, the statistical properties of all individual pixels in a detector can be characterized from a large data set and can subsequently be used to correct for imperfection of the camera (Afanasyev et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Single-particle cryo-EM using alignment by classification (ABC): the structure ofLumbricus terrestrishaemoglobin

Afanasyev

Seer-Linnemayr

Ravelli

et al. 2017

IUCrJ

Self Cite

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Single-particle cryogenic electron microscopy (cryo-EM) can now yield nearatomic resolution structures of biological complexes. However, the referencebased alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional referencefree 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of $3.8 Å is presented as an example of the use of the ABC-4D procedure.

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A posteriori correction of camera characteristics from large image data sets

Cited by 26 publications

References 23 publications

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding

Label-free single-instance protein detection in vitrified cells

Single-particle cryo-EM using alignment by classification (ABC): the structure ofLumbricus terrestrishaemoglobin

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