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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.


Related in: MedlinePlus

Rocking curve of the catalase (0, 10, 8) reflection at d = 13.7 Å, recorded in ‘rolling shutter’ mode. In all panels ϕ = 0° denotes the start of the data collection, at which point the stage is not necessarily untilted. The rotation range in all images is Δϕ = 0.36°. (a)–(h) The pixel intensities from eight successive frames as recorded by the camera, such that each node in the mesh corresponds to one pixel. (i) The profile-fitted intensities as integrated by MOSFLM, where the vertical error bars span one standard deviation. Additional rocking curves for several other spots from catalase and lysozyme are given in §3 of the supporting information.
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fig2: Rocking curve of the catalase (0, 10, 8) reflection at d = 13.7 Å, recorded in ‘rolling shutter’ mode. In all panels ϕ = 0° denotes the start of the data collection, at which point the stage is not necessarily untilted. The rotation range in all images is Δϕ = 0.36°. (a)–(h) The pixel intensities from eight successive frames as recorded by the camera, such that each node in the mesh corresponds to one pixel. (i) The profile-fitted intensities as integrated by MOSFLM, where the vertical error bars span one standard deviation. Additional rocking curves for several other spots from catalase and lysozyme are given in §3 of the supporting information.

Mentions: Continuous-rotation data sets from single crystals are collected in shutterless mode in about 10 min. The detector is constantly exposed and read out at regularly spaced intervals. This mode of operation trades detector accuracy for simplified experimental setup; in particular we find that the effects of intensity accumulation and uninterrupted sample rotation during the ∼0.1 s read-out time of the detector are negligible (Fig. 2 ▸ and §2 in the supporting information).


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)

Rocking curve of the catalase (0, 10, 8) reflection at d = 13.7 Å, recorded in ‘rolling shutter’ mode. In all panels ϕ = 0° denotes the start of the data collection, at which point the stage is not necessarily untilted. The rotation range in all images is Δϕ = 0.36°. (a)–(h) The pixel intensities from eight successive frames as recorded by the camera, such that each node in the mesh corresponds to one pixel. (i) The profile-fitted intensities as integrated by MOSFLM, where the vertical error bars span one standard deviation. Additional rocking curves for several other spots from catalase and lysozyme are given in §3 of the supporting information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Rocking curve of the catalase (0, 10, 8) reflection at d = 13.7 Å, recorded in ‘rolling shutter’ mode. In all panels ϕ = 0° denotes the start of the data collection, at which point the stage is not necessarily untilted. The rotation range in all images is Δϕ = 0.36°. (a)–(h) The pixel intensities from eight successive frames as recorded by the camera, such that each node in the mesh corresponds to one pixel. (i) The profile-fitted intensities as integrated by MOSFLM, where the vertical error bars span one standard deviation. Additional rocking curves for several other spots from catalase and lysozyme are given in §3 of the supporting information.
Mentions: Continuous-rotation data sets from single crystals are collected in shutterless mode in about 10 min. The detector is constantly exposed and read out at regularly spaced intervals. This mode of operation trades detector accuracy for simplified experimental setup; in particular we find that the effects of intensity accumulation and uninterrupted sample rotation during the ∼0.1 s read-out time of the detector are negligible (Fig. 2 ▸ and §2 in the supporting information).

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.


Related in: MedlinePlus