Approaches to altering particle distributions in cryo-electron microscopy sample preparation

Drulyte, Ieva; Johnson, Rachel M.; Hesketh, Emma L.; Hurdiss, Daniel L.; Scarff, Charlotte A.; Porav, Sebastian; Ranson, Neil A.; Muench, Stephen P.; Thompson, Rebecca

doi:10.1107/s2059798318006496

Cited by 131 publications

(123 citation statements)

References 74 publications

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“…To facilitate direct comparison, we collected tomograms from regions of matching ice thickness on grids prepared using a Vitrobot. Consistent with previous reports [36][37][38] we observed a pronounced bi-phasic distribution of sample due to clustering at the air-water interfaces when prepared on a Vitrobot (Figure 4D) among multiple areas of independently produced grids (Figure 4E). Importantly, following the same analysis and quantifications of sample grids prepared by trEM we observed a significantly more uniform distribution of protein along the z-direction (Figure 4F, G), indicating an important improvement to sample quality.…”

Section: Quality Assessment Of Cryo-em Sample Prepared By Blot-free Ssupporting

confidence: 92%

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mäeots

Lee

Nans

et al. 2020

Preprint

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Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise shortlived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

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Section: Quality Assessment Of Cryo-em Sample Prepared By Blot-free Ssupporting

confidence: 92%

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mäeots

Lee

Nans

et al. 2020

Preprint

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“…This can lead to protein denaturation but also, in almost all instances, particles are adsorbed to such interfaces and present preferential orientation due to their surface properties 17 . While supplementation of buffers with surfactants such as detergents is often employed to improve particle distribution in ice 18 , for sensitive specimens including membrane proteins this approach should be avoided. To limit the impact of preferential particle orientation, specimens can be tilted inside the microscope during data collection 19 .…”

Section: Introductionmentioning

confidence: 99%

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Uchański

Masiulis

Fischer

et al. 2019

Preprint

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Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 Å resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABA A b3 homopentameric receptor bound to its small-molecule agonist histamine.

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“…5). For cryo-EM, an aqueous sample is placed on a carbon-coated copper grid which is plunged in liquid ethane that vitrifies the water into a glass-like state to enable the sample to be seen [43,44]. Ice thickness can vary depending on the dimensions of the particle but usually range from a few nanometres to a hundred nanometres [44].…”

Section: Preprintsmentioning

confidence: 99%

“…The thicker the ice (>100 nm), the worse the resolution [45]. On the other hand, if the ice is too thin then either the sample is pushed towards the edge of the grid holes, or the sample can have a high affinity for the carbon support leaving the grid hole empty of sample and causing particle aggregation [43].…”

Section: Preprintsmentioning

confidence: 99%

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

Seneviratne¹,

Jeuken²,

Rappolt³

et al. 2020

Preprint

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Sterilisation and preservation of vesicle formulations are an important consideration for their viable manufacture for industry applications, particular those intended for medicinal use. Here we undertake an initial investigation of the stability of hybrid lipid -block copolymer vesicles to common sterilisation and preservation processes, with particular interest in how the block copolymer component might tune vesicle stability. We investigate two sizes of polybutadiene-blockpoly(ethylene oxide) polymers (PBd12-PEO11 and PBd22-PEO14) mixed with the phospholipid POPC considering the encapsulation stability of a fluorescent cargo and the colloidal stability of vesicle size distributions. We find that autoclaving and lyophilisation cause complete loss of encapsulation stability under the conditions studied here. Filtering through 200 nm pores appears to be viable for sterilisation for all vesicle compositions with comparatively low release of encapsulated cargo, even for vesicle size distributions which extend beyond the 200 nm filter pore size. Freeze-thaw of vesicles also shows promise for preservation of hybrid vesicles with high block copolymer content. We discuss the process stability of hybrid vesicles in terms of the complex mechanical interplay between bending resistance, stretching elasticity and lysis strain of these membranes and propose strategies for future work to further enhance the process stability of these vesicle formulations.

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Approaches to altering particle distributions in cryo-electron microscopy sample preparation

Abstract: This paper describes different approaches that cryo-EM users can take to improve the quality of their sample distribution and ice for high-resolution single-particle cryo-EM.

Cited by 131 publications

References 74 publications

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Hybrid Vesicle Stability under Sterilisation and Preservation Processes Used in the Manufacture of Medicinal Formulations

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