Automated batch fiducial-less tilt-series alignment in Appion using Protomo

Noble, Alex J.; Stagg, Scott M.

doi:10.1016/j.jsb.2015.10.003

Cited by 74 publications

(64 citation statements)

References 34 publications

(36 reference statements)

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“…We hypothesized that the preferred orientation was due to adsorption and orientation of the particles at the airwater interface [8] and that CHAPSO mitigated this problem by preventing adsorption at the interface. We used fiducial-less cryo-electron tomography (cryo-ET) on the singleparticle specimens to visualize particle distributions within the vitreous ice [9,10]. The results confirmed our hypothesis; the orientation bias arises from interactions of the particles with the surfaces of the ice layer.…”

Section: Introductionsupporting

confidence: 65%

“…We employed fiducial-less cryo-electron tomography (cryo-ET) on the singleparticle specimens in order to visualize the locations of the particles in the vitreous ice layer [9,10]. Tilt series of cryo-grids of the 6S-Eσ 70 complex were collected for each solution condition (Fig.…”

Section: Cryo-electron Tomography Shows That Orientation Bias Correspmentioning

confidence: 99%

“…Tilt-series were aligned using Appion-Protomo [10,24,25]. Tilt-series were first dose compensated using equation 3 in Grant, Grigorieff [26], coarsely aligned, manually fixed if necessary, refined using a set of alignment thicknesses, then the best aligned iteration was reconstructed for visual analysis using Tomo3D SIRT [27,28].…”

Section: Cryo-et Data Analysismentioning

confidence: 99%

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Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

Chen

Noble

Kang

et al. 2018

Preprint

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Preferred particle orientation presents a major challenge for many single particle cryoelectron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed to be due to adsorption of the particles at the air/water interface during cryo-EM grid preparation. To ameliorate this problem, detergents are often added to cryo-EM samples to alter the properties of the air/water interface. We have found that many bacterial transcription complexes suffer severe orientation bias when examined by cryo-EM. The addition of non-ionic detergents, such as NP-40, does not remove the orientation bias but the Zwitter-ionic detergent CHAPSO significantly broadens the particle orientation distributions, yielding isotropically uniform maps. We used cryoelectron tomography to examine the particle distribution within the ice layer of cryo-EM grid preparations of Escherichia coli 6S RNA/RNA polymerase holoenzyme particles. In the absence of CHAPSO, essentially all of the particles are located at the ice surfaces.CHAPSO at the critical micelle concentration eliminates particle absorption at the air/water interface and allows particles to randomly orient in the vitreous ice layer. We find that CHAPSO eliminates orientation bias for a wide range of bacterial transcription complexes containing E. coli or Mycobacterium tuberculosis RNA polymerases.Findings of this study confirm the presumed basis for how detergents can help remove orientation bias in cryo-EM samples and establishes CHAPSO as a useful tool to facilitate cryo-EM studies of baterial transcription complexes.3

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Section: Introductionsupporting

confidence: 65%

Section: Cryo-electron Tomography Shows That Orientation Bias Correspmentioning

confidence: 99%

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Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

Chen

Noble

Kang

et al. 2018

Preprint

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“…However, tiltseries alignment is known to be error prone due to the individual movements of gold fiducials in vitreous ice (Iancu et al 2006; Noble, & Stagg 2015). Furthermore, in addition to translational drift and radiation damage, single particles undergo out-of-plane rotational motions (Brilot et al 2012), breaking, at the subtomogram level, the central assumption of tomography that all specimen images in a tiltseries are only rotated about a common tilt axis.…”

Section: Discussionmentioning

confidence: 99%

Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography

Galaz-Montoya

Hecksel²,

Baldwin

et al. 2016

Journal of Structural Biology

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Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen grid and carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.

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“…Noble and Stagg (2015) recently described a system for automated batch processing within the Appion framework (Lander et al, 2009), based on processing with Protomo (Winkler and Taylor, 2006). Some of the chief differences from IMOD processing are: it appears to work only with data acquired with Leginon (Suloway et al, 2009); it relies on one fiducial-less alignment method, which may well be better than our patch tracking for some tilt series, but would be unsuitable for large-area data requiring correction for nonlinear changes under the beam; and the alignment time is reported to be several hours.…”

Section: Discussionmentioning

confidence: 99%

Automated tilt series alignment and tomographic reconstruction in IMOD

Mastronarde

Held

2017

Journal of Structural Biology

620

544

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Automated tomographic reconstruction is now possible in the IMOD software package, including the merging of tomograms taken around two orthogonal axes. Several developments enable the production of high-quality tomograms. When using fiducial markers for alignment, the markers to be tracked through the series are chosen automatically; if there is an excess of markers available, a well-distributed subset is selected that is most likely to track well. Marker positions are refined by applying an edge-enhancing Sobel filter, which results in a 20% improvement in alignment error for plastic-embedded samples and 10% for frozen-hydrated samples. Robust fitting, in which outlying points are given less or no weight in computing the fitting error, is used to obtain an alignment solution, so that aberrant points from the automated tracking can have little effect on the alignment. When merging two dual-axis tomograms, the alignment between them is refined from correlations between local patches; a measure of structure was developed so that patches with insufficient structure to give accurate correlations can now be excluded automatically. We have also developed a script for running all steps in the reconstruction process with a flexible mechanism for setting parameters, and we have added a user interface for batch processing of tilt series to the Etomo program in IMOD. Batch processing is fully compatible with interactive processing and can increase efficiency even when the automation is not fully successful, because users can focus their effort on the steps that require manual intervention.

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Automated batch fiducial-less tilt-series alignment in Appion using Protomo

Cited by 74 publications

References 34 publications

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography

Automated tilt series alignment and tomographic reconstruction in IMOD

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