Electron diffraction data processing with<i>DIALS</i>

Clabbers, Max T. B.; Gruene, Tim; Parkhurst, James M.; Abrahams, Jan Pieter; Waterman, David

doi:10.1107/s2059798318007726

Cited by 112 publications

(92 citation statements)

References 54 publications

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“…or DIALS(Clabbers et al 2018) and scaled with AIMLESS(Evans and Murshudov 2013). Structures were solved by molecular replacement with PHASER (McCoy et al 2007) using the WT gelsolin crystal structure (PDB ID 3FFN (Nag et al 2009)) as a search model.…”

mentioning

confidence: 99%

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties

et al. 2019

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Mutations in the gelsolin protein are responsible for a rare conformational disease known as AGel amyloidosis. Four of these mutations are hosted by the second domain of the protein (G2): D187N/Y, G167R and N184K. The impact of the latter has been so far evaluated only by studies on the isolated G2. Here we report the characterization of full-length gelsolin carrying the N184K mutation and compare the findings with those obtained on the wild type and the other variants.The crystallographic structure of the N184K variant in the Ca 2+ -free conformation shows remarkable similarities with the wild type protein. Only minimal local rearrangements can be observed and the mutant is as efficient as the wild type in severing filamentous actin. However, thermal stability of the pathological variant is compromised in the Ca 2+ -free conditions. These data suggest that the N to K substitution causes a local disruption of the H-bond network in the 2 core of the G2 domain. Such a subtle rearrangement of the connections does not lead to significant conformational changes but severely affects the stability of the protein.

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mentioning

confidence: 99%

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties

et al. 2019

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“…MicroED data were indexed, integrated, and scaled in DIALS as described (Clabbers et al, 2018; Parkhurst et al, 2016; Waterman et al, 2016; Winter et al, 2018). A resolution cutoff was applied after integration at 1.78Å, where the CC1/2 fell to a value of 0.33 (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…The diffraction distance was set to 1900mm which corresponds to a crystal to detector distance of 1853mm after taking post-column magnification into account. Camera length was calibrated using diffraction from an aluminum film prior to loading the protein grid and to check for any distortions in the diffraction (Clabbers et al, 2018, 2017).…”

Section: Methodsmentioning

confidence: 99%

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

Martynowycz

Gonen

2020

Preprint

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A method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.

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“…Taking 1.5% off the calibrated camera distance as an initial estimate resulted in an increase in error in Rfree of more than 3% ( Table 2). The camera distance can be optimized by a postrefinement process with XDS (Kabsch, 2010) and this optimization has also been recently implemented in Refmac (Murshudov et al, 2011;Clabbers et al, 2018), but the refinement likely falls into local minima (Table 2), due to the short wavelength of electrons and broader profiles in diffraction spots along the direction normal to the plate plane of the crystal (Yonekura et al, 2015). Thus, it is best to start with the optimum camera distance.…”

Section: Our Dataset 3 (200kv and Without Energy Filtration) Is Infermentioning

confidence: 99%

A new cryo-EM system for electron 3D crystallography by eEFD

Yonekura

Ishikawa

Maki-Yonekura

2019

Preprint

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A new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.

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Electron diffraction data processing withDIALS

Abstract: Adaptations to the DIALS package are described that make it a suitable choice for processing challenging continuous-rotation electron diffraction data. The results of using the extended package are presented for a case consisting of seven example data sets.

Cited by 112 publications

References 54 publications

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties

The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

A new cryo-EM system for electron 3D crystallography by eEFD

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