Atomic resolution structure determination by the cryo‐EM method MicroED

Liu, Shian; Hattne, Johan; Reyes, F.E.; Sánchez-Martínez, Silvia; Cruz, M. Jason de la; Shi, Dan; Gonen, Tamir

doi:10.1002/pro.2989

Cited by 26 publications

(15 citation statements)

References 58 publications

(121 reference statements)

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“…Diffuse X-ray scattering (Wall et al, 2014(Wall et al, , 2018 can also reveal molecular motions and lattice disorder, but methods to exploit the information contained in diffuse scattering are still scarce. Micro-electron diffraction (MicroED; Liu et al, 2017;Gruene et al, 2018;Jones et al, 2018;Nannenga & Gonen, 2018) is an emerging technique to determine high-resolution structures of macromolecules. Current procedures for diffraction data will require fine-tuning to treat MicroED data adequately.…”

Section: Resultsmentioning

confidence: 99%

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Liebschner

Afonine

Baker

et al. 2019

Acta Crystallogr D Struct Biol

4,936

3,988

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Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

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Section: Resultsmentioning

confidence: 99%

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Liebschner

Afonine

Baker

et al. 2019

Acta Crystallogr D Struct Biol

4,936

3,988

View full text Add to dashboard Cite

show abstract

“…2f). We decided to use the cryo-EM method MicroED 52,53 on the basis of its previous success in determining key structures from three other amyloid proteins: α-synuclein, tau and IAPP 23,54–56 . With this technique, an electron microscope is used to collect data in diffraction mode.…”

Section: Resultsmentioning

confidence: 99%

Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2

Guenther

Trinh

et al. 2018

Nat Struct Mol Biol

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110

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Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.

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“…MicroED employs a standard cryo electron microscope (cryoEM) in diffraction mode for data collection from 3D crystals only a few hundred nanometers thick (Figure 2A; Figure 3A) (Shi et al, 2013; Nannenga et al, 2014a, 2014b; Hattne et al, 2015; Liu et al, 2016). Such thin crystals are capable of producing measurable Bragg peaks because electrons interact with matter more strongly than X-rays.…”

Section: Resultsmentioning

confidence: 99%

Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity

Krotee

Rodríguez

Sawaya

et al. 2017

eLife

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hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19–29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15–25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19–29 S20G may serve as a model for the toxic spine of hIAPP.DOI: http://dx.doi.org/10.7554/eLife.19273.001

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Atomic resolution structure determination by the cryo‐EM method MicroED

Cited by 26 publications

References 58 publications

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2

Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity

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