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MicroED data collection and processing.

Hattne J, Reyes FE, Nannenga BL, Shi D, de la Cruz MJ, Leslie AG, Gonen T - Acta Crystallogr A Found Adv (2015)

Bottom Line: MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals.By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples.This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

View Article: PubMed Central - HTML - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.

ABSTRACT
MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

No MeSH data available.


After interacting with the sample the beam (amber rays) passes through the objective lens, which forms a diffraction pattern at the cross-section plane and an image of the sample at the image plane. Only the diffraction pattern corresponding to the image of the crystal within the selected area aperture will be visible. Several rays are omitted in these simplified illustrations and the size of the image plane is exaggerated for clarity. The scattering angle (2θ) is indicated. (a) In bright field, the image of the crystal is magnified onto the detector (yellow rays). (b) In diffraction mode, the diffraction lens is positioned to form a magnified image of the diffraction pattern (green rays) on the detector. The objective aperture at the cross-section plane is fully open. (c) Owing to the magnification of the lenses, the distance d from the sample to the physical detector is typically much smaller than the distance D to the virtual detector. The distance to the virtual detector corresponds to the sample–detector distance in a lensless measurement using e.g. X-rays.
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fig1: After interacting with the sample the beam (amber rays) passes through the objective lens, which forms a diffraction pattern at the cross-section plane and an image of the sample at the image plane. Only the diffraction pattern corresponding to the image of the crystal within the selected area aperture will be visible. Several rays are omitted in these simplified illustrations and the size of the image plane is exaggerated for clarity. The scattering angle (2θ) is indicated. (a) In bright field, the image of the crystal is magnified onto the detector (yellow rays). (b) In diffraction mode, the diffraction lens is positioned to form a magnified image of the diffraction pattern (green rays) on the detector. The objective aperture at the cross-section plane is fully open. (c) Owing to the magnification of the lenses, the distance d from the sample to the physical detector is typically much smaller than the distance D to the virtual detector. The distance to the virtual detector corresponds to the sample–detector distance in a lensless measurement using e.g. X-rays.

Mentions: Initial grid screening is done at ultra low dose rates (<10−6 e Å−2 s−1) and low magnification (∼100×) in bright field (Fig. 1 ▸a). In this configuration the entire grid can be quickly surveyed for the location and density of crystals and the thickness of the enveloping ice. Once crystal-containing regions are identified where the thickness of the ice, as judged by the contrast between the carbon support and the holes, is as thin as possible, the microscope is switched to over-focused diffraction mode (Fig. 1 ▸b). In this configuration, individual crystals can be inspected, the z height fine-tuned for eucentricity, and the center spot accurately focused by minimizing the size of the spot of the direct beam at a dose rate <10−3 e Å−2 s−1. This should be verified using the microscope’s phosphor screen as the direct beam could otherwise damage the detector. Moreover, electron hysteresis deserves special attention so that neither the spot of the direct beam nor the image shifts when switching among the various configurations. Typically, the diffraction pattern should be recorded at a dose rate of 0.01–0.05 e Å−2 s−1, with the beam configured to be approximately 5–10 µm in diameter, the objective aperture fully open and the selected area aperture set to closely match the size of the crystal. Detailed step-by-step procedures for microscope setup were published earlier (Gonen, 2013 ▸).


MicroED data collection and processing.

Hattne J, Reyes FE, Nannenga BL, Shi D, de la Cruz MJ, Leslie AG, Gonen T - Acta Crystallogr A Found Adv (2015)

After interacting with the sample the beam (amber rays) passes through the objective lens, which forms a diffraction pattern at the cross-section plane and an image of the sample at the image plane. Only the diffraction pattern corresponding to the image of the crystal within the selected area aperture will be visible. Several rays are omitted in these simplified illustrations and the size of the image plane is exaggerated for clarity. The scattering angle (2θ) is indicated. (a) In bright field, the image of the crystal is magnified onto the detector (yellow rays). (b) In diffraction mode, the diffraction lens is positioned to form a magnified image of the diffraction pattern (green rays) on the detector. The objective aperture at the cross-section plane is fully open. (c) Owing to the magnification of the lenses, the distance d from the sample to the physical detector is typically much smaller than the distance D to the virtual detector. The distance to the virtual detector corresponds to the sample–detector distance in a lensless measurement using e.g. X-rays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487423&req=5

fig1: After interacting with the sample the beam (amber rays) passes through the objective lens, which forms a diffraction pattern at the cross-section plane and an image of the sample at the image plane. Only the diffraction pattern corresponding to the image of the crystal within the selected area aperture will be visible. Several rays are omitted in these simplified illustrations and the size of the image plane is exaggerated for clarity. The scattering angle (2θ) is indicated. (a) In bright field, the image of the crystal is magnified onto the detector (yellow rays). (b) In diffraction mode, the diffraction lens is positioned to form a magnified image of the diffraction pattern (green rays) on the detector. The objective aperture at the cross-section plane is fully open. (c) Owing to the magnification of the lenses, the distance d from the sample to the physical detector is typically much smaller than the distance D to the virtual detector. The distance to the virtual detector corresponds to the sample–detector distance in a lensless measurement using e.g. X-rays.
Mentions: Initial grid screening is done at ultra low dose rates (<10−6 e Å−2 s−1) and low magnification (∼100×) in bright field (Fig. 1 ▸a). In this configuration the entire grid can be quickly surveyed for the location and density of crystals and the thickness of the enveloping ice. Once crystal-containing regions are identified where the thickness of the ice, as judged by the contrast between the carbon support and the holes, is as thin as possible, the microscope is switched to over-focused diffraction mode (Fig. 1 ▸b). In this configuration, individual crystals can be inspected, the z height fine-tuned for eucentricity, and the center spot accurately focused by minimizing the size of the spot of the direct beam at a dose rate <10−3 e Å−2 s−1. This should be verified using the microscope’s phosphor screen as the direct beam could otherwise damage the detector. Moreover, electron hysteresis deserves special attention so that neither the spot of the direct beam nor the image shifts when switching among the various configurations. Typically, the diffraction pattern should be recorded at a dose rate of 0.01–0.05 e Å−2 s−1, with the beam configured to be approximately 5–10 µm in diameter, the objective aperture fully open and the selected area aperture set to closely match the size of the crystal. Detailed step-by-step procedures for microscope setup were published earlier (Gonen, 2013 ▸).

Bottom Line: MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals.By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples.This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

View Article: PubMed Central - HTML - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.

ABSTRACT
MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.

No MeSH data available.