Solving a new R2lox protein structure by microcrystal electron diffraction

Xu, Hongyi; Lebrette, Hugo; Clabbers, Max T. B.; Zhao, Jingjing; Griese, Julia J.; Zou, Xiaodong; Högbom, Martin

doi:10.1126/sciadv.aax4621

Cited by 73 publications

(92 citation statements)

References 72 publications

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“…In each case, the use of near-ideal models also overcame potential issues with data quality that may pose barriers to phasing, including low completeness or high integration errors (Hattne et al, 2015). With continued improvements to data collection and processing, novel structures continue to be determined by MicroED (Hughes et al, 2018;Jones et al, 2018;Purdy et al, 2018;de la Cruz et al, 2017;Zhou et al, 2019;Xu et al, 2018Xu et al, , 2019. Despite these successes, caution is prudent when evaluating the influence of model bias on the final structures, particularly where the model-to-structure r.m.s.d.…”

Section: Introductionmentioning

confidence: 99%

Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER

Richards

Millán

Miao

et al. 2020

Acta Cryst Sect D Struct Biol

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Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.

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Section: Introductionmentioning

confidence: 99%

Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER

Richards

Millán

Miao

et al. 2020

Acta Cryst Sect D Struct Biol

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“…Since 2013, when the first protein structure was determined by the microcrystal electron diffraction (MicroED) technique (Shi et al, 2013), 3D macromolecular crystallography with electrons has undergone significant developments in data collection and data analysis (Nannenga et al, 2014;van Genderen et al, 2016;Clabbers et al, 2017;Gruene et al, 2018;Hattne et al, 2019;Bücker et al, 2020), and a number of new protein and peptide structures have been solved (Rodriguez et al, 2015;de la Cruz et al, 2017;Xu et al, 2019). For a typical ED data collection experiment, crystals in solution are pipetted onto a transmission electron microscopy (TEM) grid, blotted to remove excess solution, and vitrified in liquid ethane or nitrogen.…”

Section: Introductionmentioning

confidence: 99%

“…Protein and peptide crystals below ~0.5-µm thickness have successfully been used for structure determination (Rodriguez et al, 2015;Sawaya et al, 2016;Clabbers et al, 2017;Xu et al, 2019). Bigger crystals can be broken up to smaller fragments by mechanical force (e.g.…”

Section: Introductionmentioning

confidence: 99%

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

Polovinkin

Khakurel

Babiak

et al. 2020

Preprint

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AbstractElectron crystallography of sub-micron sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realising 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix, surrounding the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, we experimentally verified the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB) can be used to remove excess host lipidic mesophase matrix, and then thin the crystals to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic mesophase of monoolein were used as a model system. LCP from a part of a 50-μm thick crystal, which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210-nm thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope (cryo-EM), and a nanovolume of ∼1400×1400×210 nm3 of the BR lamella was exposed to 200-kV electrons at a fluence of ∼0.06 e−/Å2. The resulting electron diffraction peaks were detected beyond 2.7-Å resolution (with mean signal-to-noise ratio <I/σ(I)> of >7) by a CMOS-based Ceta 16M camera. The results demonstrate, that cryo-FIB milling produces high quality lamellae from crystals grown in lipidic mesophases, and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media.SynopsisElectron diffraction experiments on crystals of membrane proteins grown in lipidic mesophases have not been possible due to a thick layer of viscous crystallisation medium around the crystals. Here we show that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can remove the viscous layer, and demonstrate high-quality electron diffraction on a FIB-milled lamella of a bacteriorhodopsin 3D crystal.

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“…Microcrystal electron diffraction (MicroED) is an electron cryo-microscopy (cryoEM) method for determining atomic resolution structures from protein microcrystals (Brent L Nannenga et al, 2014; Brent L. Nannenga et al, 2014; Shi et al, 2013). MicroED has been used to solve novel protein and small molecule pharmaceutical crystal structures (de la Cruz et al, 2017; Gruene et al, 2018; Jones et al, 2018; Rodriguez et al, 2015; Xu et al, 2019). Previous ligand-protein interactions determined using MicroED relied on co-crystallization or incubation of the protein and ligand in crystallization drops prior to applying the crystals to the grid (Clabbers et al, 2020; Purdy et al, 2018; Seidler et al, 2018).…”

Section: Introductionmentioning

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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Solving a new R2lox protein structure by microcrystal electron diffraction

Cited by 73 publications

References 72 publications

Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER

Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER

Demonstration of electron diffraction from membrane protein crystals grown in a lipidic mesophase after lamella preparation by focused ion beam milling at cryogenic temperatures

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

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