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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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The vitrified section process affects the specimen geometry in various ways: Damage by the cutting knife such as compression or crevasses and its effect to macromolecular structures has to be carefully analyzed before volume averaging procedures can be applied. Large-scale distortions and compression along the cutting directions (red arrows) affect membrane organelles, including microtubules cross-section and shape (MT in a, c). a 2-D projection micrograph through a vitrified section of 3T3 cell showing multiple membrane vesicles and some microtubules towards the center. Most organelles appear compressed along the cutting direction. b 2-D projection micrograph of a vitrified section through S. pombe cells revealing the typical distortions encountered during vitrified sectioning such as knife marks and crevasses. c 21-nm tomographic slice through a cross-section of a Trypanosoma brucei cell reveals the spectacular microtubule-based cytoskeleton of this single-cell organism around the edge. Most of the microtubules appear squeezed roughly along the direction of the knife marks, though the compression is not uniform (see also Höög et al. 2012)
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Fig3: The vitrified section process affects the specimen geometry in various ways: Damage by the cutting knife such as compression or crevasses and its effect to macromolecular structures has to be carefully analyzed before volume averaging procedures can be applied. Large-scale distortions and compression along the cutting directions (red arrows) affect membrane organelles, including microtubules cross-section and shape (MT in a, c). a 2-D projection micrograph through a vitrified section of 3T3 cell showing multiple membrane vesicles and some microtubules towards the center. Most organelles appear compressed along the cutting direction. b 2-D projection micrograph of a vitrified section through S. pombe cells revealing the typical distortions encountered during vitrified sectioning such as knife marks and crevasses. c 21-nm tomographic slice through a cross-section of a Trypanosoma brucei cell reveals the spectacular microtubule-based cytoskeleton of this single-cell organism around the edge. Most of the microtubules appear squeezed roughly along the direction of the knife marks, though the compression is not uniform (see also Höög 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)

The vitrified section process affects the specimen geometry in various ways: Damage by the cutting knife such as compression or crevasses and its effect to macromolecular structures has to be carefully analyzed before volume averaging procedures can be applied. Large-scale distortions and compression along the cutting directions (red arrows) affect membrane organelles, including microtubules cross-section and shape (MT in a, c). a 2-D projection micrograph through a vitrified section of 3T3 cell showing multiple membrane vesicles and some microtubules towards the center. Most organelles appear compressed along the cutting direction. b 2-D projection micrograph of a vitrified section through S. pombe cells revealing the typical distortions encountered during vitrified sectioning such as knife marks and crevasses. c 21-nm tomographic slice through a cross-section of a Trypanosoma brucei cell reveals the spectacular microtubule-based cytoskeleton of this single-cell organism around the edge. Most of the microtubules appear squeezed roughly along the direction of the knife marks, though the compression is not uniform (see also Höög et al. 2012)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3927062&req=5

Fig3: The vitrified section process affects the specimen geometry in various ways: Damage by the cutting knife such as compression or crevasses and its effect to macromolecular structures has to be carefully analyzed before volume averaging procedures can be applied. Large-scale distortions and compression along the cutting directions (red arrows) affect membrane organelles, including microtubules cross-section and shape (MT in a, c). a 2-D projection micrograph through a vitrified section of 3T3 cell showing multiple membrane vesicles and some microtubules towards the center. Most organelles appear compressed along the cutting direction. b 2-D projection micrograph of a vitrified section through S. pombe cells revealing the typical distortions encountered during vitrified sectioning such as knife marks and crevasses. c 21-nm tomographic slice through a cross-section of a Trypanosoma brucei cell reveals the spectacular microtubule-based cytoskeleton of this single-cell organism around the edge. Most of the microtubules appear squeezed roughly along the direction of the knife marks, though the compression is not uniform (see also Höög 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