Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size

Martynowycz, Michael W.; Wei, Zhao; Hattne, Johan; Jensen, Grant J.; Gonen, Tamir

doi:10.1016/j.str.2018.12.003

Cited by 63 publications

(82 citation statements)

References 22 publications

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“…We applied the GIS platinum layer in the mapping position to prevent shadowing of the platinum layer and opened the GIS valve at 12mm rather than 7mm in order to have a slower, more controlled deposition. Crystals were identified in the SEM and FIB, brought to eucentric height, and milled using cleaning cross sections in steps into lamellae until a thickness of ~200nm as described (Duyvesteyn et al, 2018; Michael W. Martynowycz et al, 2019). The final polishing step used a gallium beam current of 10pA to remove the last few nm of crystal from the lamellae (Michael W Martynowycz et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

“…MicroED data collection was performed very similarly to previous experiments (Hattne et al, 2019, 2018, 2015; Jason de la Cruz et al, 2019; Martynowycz et al, 2020; Michael W Martynowycz et al, 2019; Michael W. Martynowycz et al, 2019). Grids with milled crystals were transferred to a Thermo-Fisher Talos Arctica transmission electron microscope after rotating the grids by 90°.…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Martynowycz

Gonen

2020

Preprint

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“…vigorous pipetting, sonication, vortexing with beads and etc.) (de la Cruz et al, 2017) or can be thinned in a more reproducible and reliable way to a desired size by focused ion beam milling under cryogenic conditions (cryo-FIB) (Duyvesteyn et al, 2018;Zhou et al, 2019;Martynowycz et al, 2019a). The latter technique has been inherited from material science (Giannuzzi & Stevie, 1999) and cellular cryo-electron tomography (cryo-ET) (Marko et al, 2007;.…”

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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“…These crystals tend to be more delicate and do not survive the harsh sonication/vortexing treatment so new sample preparation methods are required. Recently, the use of a Focused-Ion Beam on a Scanning Electron Microscope (FIB-SEM) was demonstrated as a viable sample preparation technique for MicroED (Duyvesteyn et al, 2018;Martynowycz et al, 2019aMartynowycz et al, , 2019b (Figure 1). With this method, an SEM is used to identify crystals and a FIB is used to thin the crystals to <500nm thickness creating the ideal samples for MicroED.…”

Section: Introductionmentioning

confidence: 99%

MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel

Martynowycz

Khan

Hattne

et al. 2020

Preprint

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A near-atomic resolution structure of the mouse voltage dependent anion channel (mVDAC) is determined by combining cryogenic focused ion-beam (FIB) milling and microcrystal electron diffraction (MicroED). The crystals were grown in a viscous modified bicelle suspension which limited their size and made them unsuitable for conventional X-ray crystallography. Individual thin, plate-like crystals were identified using scanning electron microscopy (SEM) and focused ion-beam (FIB) imaging at high magnification. Three crystals were milled into thin lamellae. MicroED data were collected from each lamellae and merged to increase completeness. Unmodelled densities were observed between protein monomers, suggesting the presence of lipids that likely mediate crystal contacts. This work demonstrates the utility of milling membrane protein microcrystals grown in viscous media using a focused ion-beam for subsequent structure determination by MicroED for samples that are not otherwise tractable by other crystallographic methods. To our knowledge, the structure presented here is the first of a membrane protein crystallized in a lipid matrix and solved by MicroED.

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Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size

Cited by 63 publications

References 22 publications

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

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

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

MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel

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