Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Myasnikov, Alexander G.; Afonina, Zhanna A.; Klaholz, Bruno P.

doi:10.1016/j.ultramic.2012.12.009

Cited by 17 publications

(10 citation statements)

References 47 publications

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“…1a ). The electron optical density of a complex is proportional to its electrostatic potential, which is determined by its atomic structure [ 15 , 19 ]. For each complex, density maps are generated at 4 nm resolution and with voxel size of 1 nm using the PDB2VOL program of the Situs2.0 package [ 20 ].…”

Section: Methodsmentioning

confidence: 99%

Simulating cryo electron tomograms of crowded cell cytoplasm for assessment of automated particle picking

Pei

Frazier

et al. 2016

BMC Bioinformatics

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BackgroundCryo-electron tomography is an important tool to study structures of macromolecular complexes in close to native states. A whole cell cryo electron tomogram contains structural information of all its macromolecular complexes. However, extracting this information remains challenging, and relies on sophisticated image processing, in particular for template-free particle extraction, classification and averaging. To develop these methods it is crucial to realistically simulate tomograms of crowded cellular environments, which can then serve as ground truth models for assessing and optimizing methods for detection of complexes in cell tomograms.ResultsWe present a framework to generate crowded mixtures of macromolecular complexes for realistically simulating cryo electron tomograms including noise and image distortions due to the missing-wedge effects. Simulated tomograms are then used for assessing the template-free Difference-of-Gaussian (DoG) particle-picking method to detect complexes of different shapes and sizes under various crowding and noise levels. We identified DoG parameter settings that maximize precision and recall for detecting particles over a wide range of sizes and shapes. We observed that medium sized DoG scaling factors showed the overall best performance. To further improve performance, we propose a combination strategy for integrating results from multiple parameter settings. With increasing macromolecular crowding levels, the precision of particle picking remained relatively high, while the recall was dramatically reduced, which limits the detection of sufficient copy numbers of complexes in a crowded environment. Over a wide range of increasing noise levels, the DoG particle picking performance remained stable, but dramatically reduced beyond a specific noise threshold.ConclusionsAutomatic and reference-free particle picking is an important first step in a visual proteomics analysis of cell tomograms. However, cell cytoplasm is highly crowded, which makes particle detection challenging. It is therefore important to test particle-picking methods in a realistic crowded setting. Here, we present a framework for simulating tomograms of cellular environments at high crowding levels and assess the DoG particle picking method. We determined optimal parameter settings to maximize the performance of the DoG particle-picking method.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1283-3) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Simulating cryo electron tomograms of crowded cell cytoplasm for assessment of automated particle picking

Pei

Frazier

et al. 2016

BMC Bioinformatics

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“…The sub-tomogram averaging largely removed missing wedge effects because of the different particle orientations. To reduce distortions in the reconstruction we also used dual-tilt series data collection (a combination of two single-tilt series collected separately on the same area of the specimen with an inplane specimen rotation of 90°), a technique which significantly improves the tomogram quality also for single particle reconstructions 26 . Dual-tilt tomograms were used for particle identification and orientation search for sub-tomogram averaging applied to the first tilt series.…”

Section: Methodsmentioning

confidence: 99%

“…Dual-tilt tomograms were used for particle identification and orientation search for sub-tomogram averaging applied to the first tilt series. Dual-tilt data were collected on CCD camera with a defocus of À 3 mm using an acceleration voltage of 150 kV and a magnification of 39,000 (3.1 Å pixel À 1 ) with a cumulative electron dose not exceeding 60 ē Å À 2 , with fine angular increments of 1.5°with maximum tilt angle of ±70°for the first tilt series, and 3°increments with ±60 maximum tilt for the second tilt series (larger angular sampling in the second tilt series was shown to be sufficient for improving the overall tomogram reconstruction and allows reducing the dose 26 ). In total, about 90 wheat germ polysomes were reconstructed from single-tilt (CMOS: 7 tomograms at 150 kV, 10 tomograms at 300 kV, CCD: 60 tomograms at 150 kV) and 8 dual-tilt tomograms (each tomogram contained several polysomes).…”

Section: Methodsmentioning

confidence: 99%

The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes

et al. 2014

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During protein synthesis, several ribosomes bind to a single messenger RNA (mRNA) forming large macromolecular assemblies called polyribosomes. Here we report the detailed molecular structure of a 100 MDa eukaryotic poly-ribosome complex derived from cryo electron tomography, sub-tomogram averaging and pseudo-atomic modelling by crystal structure fitting. The structure allowed the visualization of the three functional parts of the polysome assembly, the central core region that forms a rather compact left-handed supramolecular helix, and the more open regions that harbour the initiation and termination sites at either ends. The helical region forms a continuous mRNA channel where the mRNA strand bridges neighbouring exit and entry sites of the ribosomes and prevents mRNA looping between ribosomes. This structure provides unprecedented insights into protein-and RNAmediated inter-ribosome contacts that involve conserved sites through 40S subunits and long protruding RNA expansion segments, suggesting a role in stabilizing the overall polyribosomal assembly.

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“…The impact of direct electron detectors is illustrated by the improved resolution of the tomograms (Figure ). In the future, contrast‐increasing phase plates may be helpful to address more of the molecular details within polysomes, possibly in combination with double tilt cryo‐ET which provides 3D reconstructions with less reconstruction artefacts (Myasnikov et al., ; one of the first examples of single particle cryo‐ET together with some other examples: Dudkina et al., ; Murata et al., ; Wang et al., ).…”

Section: Cryo‐et Analysis and Sub‐tomogram Averagingmentioning

confidence: 99%

“…Recently, stunning insights into cellular sub‐structures have been obtained (Hagen et al., ; Nans et al., ; Chang et al., ; Irobalieva et al., ; Kosinski et al., ; Lin et al., ; Mahamid et al., ), achieving in a first case even side‐chain resolution (Schur et al., ) using optimised cryo‐ET, sub‐tomogram averaging and a new dose‐symmetric tilt acquisition scheme that preserves high‐resolution data more isotropically (Hagen et al., ). We used cryo‐ET in combination with sub‐tomogram averaging and molecular modelling to address the supramolecular organisation of eukaryotic polyribosomes which can form large macromolecular assemblies (Brandt et al., ; Myasnikov et al., ; Afonina et al., ; Myasnikov et al., ; Afonina et al., ). This allowed deriving the 3D structure of one of the largest asymmetric complexes to date (∼100 MDa, comprising over 20 ribosomes on the same mRNA molecule; Myasnikov et al., ).…”

Section: Cryo‐et Analysis and Sub‐tomogram Averagingmentioning

confidence: 99%

The integrative role of cryo electron microscopy in molecular and cellular structural biology

Orlov

Myasnikov

Andronov

et al. 2016

Biology of the Cell

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After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo-EM) is now heading off at unprecedented speed towards high-resolution analysis of biological objects of various sizes. This 'revolution in resolution' is happening largely thanks to new developments of new-generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo-EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo-EM in synergy with other methods such as X-ray crystallography, fluorescence imaging or focussed-ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi-scale and multi-resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.

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Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Cited by 17 publications

References 47 publications

Simulating cryo electron tomograms of crowded cell cytoplasm for assessment of automated particle picking

Simulating cryo electron tomograms of crowded cell cytoplasm for assessment of automated particle picking

The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes

The integrative role of cryo electron microscopy in molecular and cellular structural biology

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