Analysis of Global and Site-Specific Radiation Damage in Cryo-EM

Hattne, Johan; Shi, Dan; Glynn, Calina; Zee, Chih-Te; Gallagher-Jones, Marcus; Martynowycz, Michael W.; Rodríguez, José A.; Gonen, Tamir

doi:10.1016/j.str.2018.03.021

Cited by 162 publications

(141 citation statements)

References 47 publications

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“…Although the exposure per illuminated region in nanoEDT is considerably higher than in microED, the observed impacts of its higher exposure on the final structure determined by nanoEDT are more consistent with a conventional microED exposure 30 . To evaluate the impact of electron exposure during nanoEDT data collection, we compared our nanoEDT structure of the OsPYL/RCAR5 peptide to those determined by MicroED under various exposures at cryogenic conditions to a 300 kV electron beam.…”

Section: Resultssupporting

confidence: 58%

Atomic structures determined from digitally defined nanocrystalline regions

Gallagher-Jones

Bustillo

Ophus

et al. 2019

Preprint

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AbstractNanocrystallography has transformed our ability to interrogate the atomic structures of proteins, peptides, organic molecules and materials. By probing atomic level details in ordered sub-10 nm regions of nanocrystals, approaches in scanning nanobeam electron diffraction extend the reach of nanocrystallography and mitigate the need for diffraction from large portions of one or more crystals. We now apply scanning nanobeam electron diffraction to determine atomic structures from digitally defined regions of beam-sensitive peptide nanocrystals. Using a direct electron detector, we record thousands of sparse diffraction patterns over multiple crystal orientations. We assign each pattern to a specific location on a single nanocrystal with axial, lateral and angular coordinates. This approach yields a collection of patterns that represent a tilt series across an angular wedge of reciprocal space: a scanning nanobeam diffraction tomogram. From this diffraction tomogram, we can digitally extract intensities from any desired region of a scan in real or diffraction space, exclusive of all other scanned points. Intensities from multiple regions of a crystal or from multiple crystals can be merged to increase data completeness and mitigate missing wedges. Merged intensities from digitally defined regions of two crystals of a segment from the OsPYL/RCAR5 protein produce fragment-based ab-initio solutions that can be refined to atomic resolution, analogous to structures determined by selected area electron diffraction. In allowing atomic structures to now be determined from digitally outlined regions of a nanocrystal, scanning nanobeam diffraction tomography breaks new ground in nanocrystallography.

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Section: Resultssupporting

confidence: 58%

Atomic structures determined from digitally defined nanocrystalline regions

Gallagher-Jones

Bustillo

Ophus

et al. 2019

Preprint

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“…Both of these methods of LCP sample preparation ultimately produced quality MicroED data and density maps and models (Table 1; Fig 3). When compared to other Proteinase K structures determined by MicroED (15,16,19), both of the LCP-proteinase K structures in this work showed similar levels of data quality (e.g. resolution, R-factors), indicating that the processing methods used did not greatly affect the crystal quality of the microcrystals.…”

Section: Microed Analysis Of Treated Lcp Samplesmentioning

confidence: 53%

“…Here, we report the first MicroED structures of a model protein, Proteinase K, that has been embedded in LCP. This sample was chosen because it has served as a model sample for both MicroED and new LCP-based serial crystallography methods (13)(14)(15)(16). By treating the LCP samples with different reagents to lower the viscosity of the LCP samples, we identified two strategies -dilution using MPD and treatment of the sample with lipasethat led to high-quality MicroED samples.…”

Section: Introductionmentioning

confidence: 99%

Structure determination from lipidic cubic phase embedded microcrystals by MicroED

Zhu

Jing

et al. 2019

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The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. Because crystals grown in LCP can be limited in size, improved techniques for structure determination from these small crystals are important. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-TEM to collect electron diffraction data and determine high-resolution structures from very thin micro and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP. Proteinase K in LCP was used as a model system, and several LCP sample preparation strategies were tested. Among these, treatment with 2-Methyl-2,4-pentanediol (MPD) and lipase were both able to reduce the viscosity of the LCP and produce quality cryo-EM grids with welldiffracting crystals. These results set the stage for the use of MicroED to analyze other microcrystalline samples grown in LCP.

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“…The reduced density may be due to the enhanced sensitivity of acidic side chains to electron radiation and subsequent damage [27], [28], which is more prominent at 200 keV compared to apoferritin structures resolved at 300 keV [5], [9]. Other residues, such as polar (Asn98, Gln112) and aromatic (Trp93) amino acids are well resolved independent of contour level.…”

Section: Textmentioning

confidence: 99%

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV “screening microscope”

Hamdi

Tüting

Semchonok

et al. 2019

Preprint

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Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC=0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device. Data were collected using a compact, two-lens illumination system with a constant power objective lens, without the use of energy filters or aberration correctors. Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (x-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function Highlights • The 2.7 Å structure of mouse apoferritin was solved using a 200 keV screening cryo-microscope • The apoferritin reconstruction was resolved without an energy filter, aberration correctors, or constant-power condenser lenses • Comparison to available crystallographic and cryo-EM structures from highend cryo-microscopes demonstrates consistency in resolved water molecules, metals and side chain orientations • Although radiation damage is more prominent at 200 keV compared to 300 keV, this type of instrumentation is more accessible to research laboratories due to its compactness and simplicity

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Analysis of Global and Site-Specific Radiation Damage in Cryo-EM

Cited by 162 publications

References 47 publications

Atomic structures determined from digitally defined nanocrystalline regions

Atomic structures determined from digitally defined nanocrystalline regions

Structure determination from lipidic cubic phase embedded microcrystals by MicroED

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from 200 keV “screening microscope”

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