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High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.

Hoenger A - Protoplasma (2014)

Bottom Line: Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern.On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data.We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA, hoenger@colorado.edu.

ABSTRACT
Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ± 60 and ± 70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1-2° or less and a tilt range of ± 60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.

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Related in: MedlinePlus

Subvolume averaging from tomograms of plunge-frozen, regular arrays within the unique Giardia lamblia cytoskeleton. a Microtome-based block-face scanning-EM imaging of a Giardia trophozoite reveals sufficient resolution to identify flagella, nuclei (N), the median body (MB), and ventral disc (VD). These arrays constitute excellent test grounds for our labeling experiments as they allow averaging of subvolume elements and calculating difference maps for a precise detection of clonable labels. a Tomographic 20-nm thick X-Y slice of the Giardia ventral disc (green organelle in the Giardia cell shown in (c, d) at the level of the microtubule arrays. c Tomographic 20-nm thick X-Z slice of the Giardia ventral disc showing microtubules and associated microribbons end-on. d, e 3-D reconstruction of the microtubule-microribbon complex of the Giardia ventral disc. d End-on view (left panel towards the microtubule plus-end) and side view (right panel) of a grand average over 4,700 individual tomographic subvolumes. MT protofilaments are numbered, starting at the position of the seam. The largest associated densities are called side-arms (green). Currently, we do not know how many individual protein domains are within this structure. ePanels top to bottom show cross-sections at corresponding position in the 3-D map marked by lines of the following color: red (microribbons), yellow (upper microtubule protofilaments), and cyan (microtubule lumen and side arm densities). The cross-section through the microribbons reveals a distinct 16-nm repeat, corresponding to two consecutive αβ − tubulin dimers along a protofilament. Side-arms repeat in register with the tubulin dimer repeat. The 3-D map still suffers from a missing cone of data (see also Fig. 4), demonstrated by the clear separation of protofilaments horizontally, but not vertically (for further details see Schwartz et al. 2012)
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Fig1: Subvolume averaging from tomograms of plunge-frozen, regular arrays within the unique Giardia lamblia cytoskeleton. a Microtome-based block-face scanning-EM imaging of a Giardia trophozoite reveals sufficient resolution to identify flagella, nuclei (N), the median body (MB), and ventral disc (VD). These arrays constitute excellent test grounds for our labeling experiments as they allow averaging of subvolume elements and calculating difference maps for a precise detection of clonable labels. a Tomographic 20-nm thick X-Y slice of the Giardia ventral disc (green organelle in the Giardia cell shown in (c, d) at the level of the microtubule arrays. c Tomographic 20-nm thick X-Z slice of the Giardia ventral disc showing microtubules and associated microribbons end-on. d, e 3-D reconstruction of the microtubule-microribbon complex of the Giardia ventral disc. d End-on view (left panel towards the microtubule plus-end) and side view (right panel) of a grand average over 4,700 individual tomographic subvolumes. MT protofilaments are numbered, starting at the position of the seam. The largest associated densities are called side-arms (green). Currently, we do not know how many individual protein domains are within this structure. ePanels top to bottom show cross-sections at corresponding position in the 3-D map marked by lines of the following color: red (microribbons), yellow (upper microtubule protofilaments), and cyan (microtubule lumen and side arm densities). The cross-section through the microribbons reveals a distinct 16-nm repeat, corresponding to two consecutive αβ − tubulin dimers along a protofilament. Side-arms repeat in register with the tubulin dimer repeat. The 3-D map still suffers from a missing cone of data (see also Fig. 4), demonstrated by the clear separation of protofilaments horizontally, but not vertically (for further details see Schwartz et al. 2012)

Mentions: The spectacular success of 3-D reconstructions by cryo-EM and averaging procedures should not make us forget the fact that the 3-D architecture of the vast majority of biological structures cannot be subjected to averaging methods. The larger a macromolecular assembly or cell organelle is, the higher the probability of intrinsic flexibility, and the lower are the chances of structural identity between multiple copies (e.g., Figs. 1, 2, and 3). If averaging and/or alignment procedures of particles fail, the only way to obtain 3-D data is cryo-electron tomography (cryo-ET; Medalia et al. 2002; Beck et al. 2004, reviewed in Steven and Aebi 2003; Lucic et al. 2005). Today, high-resolution cryo-ET is probably the most active and fastest emerging discipline in structural cell biology as it can be applied to large macromolecular assemblies, cellular organelles, or even entire tissues. By omitting the need for any kind of averaging procedures, at least during the initial round of 3-D reconstruction, tomography can be applied to any specimen that is thin enough to get an electron beam through. Intact bacterial cells such as Caulobacter crescentus (e.g., see Briegel et al. 2006, 2009; multiple examples reviewed in Gan and Jensen 2012), or flat areas of eukaryotic cells (e.g., fibroblast peripheries; Dictyostelium: Medalia et al. 2002) may be suitable for direct imaging. All other cellular specimens have to be treated by vitrified sectioning (e.g., see McDowall et al. 1983; Al-Amoudi et al. 2004a; Dubochet et al. 2007; Bouchet-Marquis and Hoenger 2011), or by focused ion-beam milling in a dual-beam cryo-scanning electron microscope (reviewed in Lucic et al. 2013).Fig. 1


High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.

Hoenger A - Protoplasma (2014)

Subvolume averaging from tomograms of plunge-frozen, regular arrays within the unique Giardia lamblia cytoskeleton. a Microtome-based block-face scanning-EM imaging of a Giardia trophozoite reveals sufficient resolution to identify flagella, nuclei (N), the median body (MB), and ventral disc (VD). These arrays constitute excellent test grounds for our labeling experiments as they allow averaging of subvolume elements and calculating difference maps for a precise detection of clonable labels. a Tomographic 20-nm thick X-Y slice of the Giardia ventral disc (green organelle in the Giardia cell shown in (c, d) at the level of the microtubule arrays. c Tomographic 20-nm thick X-Z slice of the Giardia ventral disc showing microtubules and associated microribbons end-on. d, e 3-D reconstruction of the microtubule-microribbon complex of the Giardia ventral disc. d End-on view (left panel towards the microtubule plus-end) and side view (right panel) of a grand average over 4,700 individual tomographic subvolumes. MT protofilaments are numbered, starting at the position of the seam. The largest associated densities are called side-arms (green). Currently, we do not know how many individual protein domains are within this structure. ePanels top to bottom show cross-sections at corresponding position in the 3-D map marked by lines of the following color: red (microribbons), yellow (upper microtubule protofilaments), and cyan (microtubule lumen and side arm densities). The cross-section through the microribbons reveals a distinct 16-nm repeat, corresponding to two consecutive αβ − tubulin dimers along a protofilament. Side-arms repeat in register with the tubulin dimer repeat. The 3-D map still suffers from a missing cone of data (see also Fig. 4), demonstrated by the clear separation of protofilaments horizontally, but not vertically (for further details see Schwartz et al. 2012)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927062&req=5

Fig1: Subvolume averaging from tomograms of plunge-frozen, regular arrays within the unique Giardia lamblia cytoskeleton. a Microtome-based block-face scanning-EM imaging of a Giardia trophozoite reveals sufficient resolution to identify flagella, nuclei (N), the median body (MB), and ventral disc (VD). These arrays constitute excellent test grounds for our labeling experiments as they allow averaging of subvolume elements and calculating difference maps for a precise detection of clonable labels. a Tomographic 20-nm thick X-Y slice of the Giardia ventral disc (green organelle in the Giardia cell shown in (c, d) at the level of the microtubule arrays. c Tomographic 20-nm thick X-Z slice of the Giardia ventral disc showing microtubules and associated microribbons end-on. d, e 3-D reconstruction of the microtubule-microribbon complex of the Giardia ventral disc. d End-on view (left panel towards the microtubule plus-end) and side view (right panel) of a grand average over 4,700 individual tomographic subvolumes. MT protofilaments are numbered, starting at the position of the seam. The largest associated densities are called side-arms (green). Currently, we do not know how many individual protein domains are within this structure. ePanels top to bottom show cross-sections at corresponding position in the 3-D map marked by lines of the following color: red (microribbons), yellow (upper microtubule protofilaments), and cyan (microtubule lumen and side arm densities). The cross-section through the microribbons reveals a distinct 16-nm repeat, corresponding to two consecutive αβ − tubulin dimers along a protofilament. Side-arms repeat in register with the tubulin dimer repeat. The 3-D map still suffers from a missing cone of data (see also Fig. 4), demonstrated by the clear separation of protofilaments horizontally, but not vertically (for further details see Schwartz et al. 2012)
Mentions: The spectacular success of 3-D reconstructions by cryo-EM and averaging procedures should not make us forget the fact that the 3-D architecture of the vast majority of biological structures cannot be subjected to averaging methods. The larger a macromolecular assembly or cell organelle is, the higher the probability of intrinsic flexibility, and the lower are the chances of structural identity between multiple copies (e.g., Figs. 1, 2, and 3). If averaging and/or alignment procedures of particles fail, the only way to obtain 3-D data is cryo-electron tomography (cryo-ET; Medalia et al. 2002; Beck et al. 2004, reviewed in Steven and Aebi 2003; Lucic et al. 2005). Today, high-resolution cryo-ET is probably the most active and fastest emerging discipline in structural cell biology as it can be applied to large macromolecular assemblies, cellular organelles, or even entire tissues. By omitting the need for any kind of averaging procedures, at least during the initial round of 3-D reconstruction, tomography can be applied to any specimen that is thin enough to get an electron beam through. Intact bacterial cells such as Caulobacter crescentus (e.g., see Briegel et al. 2006, 2009; multiple examples reviewed in Gan and Jensen 2012), or flat areas of eukaryotic cells (e.g., fibroblast peripheries; Dictyostelium: Medalia et al. 2002) may be suitable for direct imaging. All other cellular specimens have to be treated by vitrified sectioning (e.g., see McDowall et al. 1983; Al-Amoudi et al. 2004a; Dubochet et al. 2007; Bouchet-Marquis and Hoenger 2011), or by focused ion-beam milling in a dual-beam cryo-scanning electron microscope (reviewed in Lucic et al. 2013).Fig. 1

Bottom Line: Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern.On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data.We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA, hoenger@colorado.edu.

ABSTRACT
Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ± 60 and ± 70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1-2° or less and a tilt range of ± 60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.

Show MeSH
Related in: MedlinePlus