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

Clabbers

Unge

et al. 2021

Preprint

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The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several EM grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion-beam scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages but the quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods.

Section: Resultsmentioning

confidence: 99%

Benchmarking ideal sample thickness in cryo-EM using MicroED

Clabbers

Unge

et al. 2021

Preprint

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“…With every step the grid preparation improved and the resulting data that was obtained likewise improved leading to high quality data (Table 1) that had high signal to noise ratio (Supplementary Figure 9). We first attempted to prepare grids for MicroED by depositing an entire crystal drop onto the TEM grid and gently blotting from the opposite side as described (Martynowycz and Gonen, 2020). These attempts resulted in samples that were completely opaque and could not be penetrated by the electron beam.…”

Section: Resultsmentioning

confidence: 99%

“…The sample chamber of the vitrification robot was held at 90% humidity. This mixture was allowed to incubate for 20s, and was then gently blotted from the back and immediately vitrified in liquid ethane as described (Martynowycz and Gonen, 2020). Grids were transferred and stored in liquid nitrogen prior to further investigation.…”

mentioning

confidence: 99%

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

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.

“…Fisher Aquilos dual beam FIB SEM as described (32,38,40). A thick layer of sputter coated platinum approximately nm thick was deposited on the grids in order to protect the samples from the damaging gallium beam.…”

Section: Main Textmentioning

confidence: 99%

“…Lamellae sites were identified in the SEM map and crystals were verified by imaging at grazing incidence in the FIB. Select crystals were milled as described (22,32,33,38,40,41). Briefly, crystals were adjusted to eucentric height.…”

Section: Main Textmentioning

confidence: 99%

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

Shiriaeva

et al. 2020

Preprint

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G Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods.One sentence summaryFIB milled LCP-GPCR structure determined by MicroED