Routine determination of ice thickness for cryo-EM grids

Rice, William J.; Cheng, Anchi; Noble, Alex J.; Kim, Laura Y.; Carragher, Bridget; Potter, Clinton S.

doi:10.1016/j.jsb.2018.06.007

Cited by 130 publications

(141 citation statements)

References 37 publications

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“…Attenuation constants for scattering losses were determined as described by (Feja and Aebi 1999). Scattering lengths that we found were 283 nm for total loss (with a 100 μm aperture) and 314 nm (no aperture (Rice et al 2018); see Supplemental Figure 2). The attenuation in SNR with sample thickness, T (Fig.…”

Section: Methodsmentioning

confidence: 94%

Label-free single-instance protein detection in vitrified cells

Choi

Lippincott‐Schwartz

et al. 2020

Preprint

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A general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of imagescalculated on the basis of known structural data-as search templates to detect targets as small as the "head" domain (350 kDa) of the ribosome's small subunit in single-tilt electron cryomicrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryoelectron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling. Main:Finding molecular components in intact cells has driven the development of biological imaging techniques, some of which use specific labels, such as fluorescent antibodies, while others rely on a target's intrinsic properties such as its shape (Oliver 1973) and structure (Rickgauer, Grigorieff, and Denk 2017). Labelling is frequently incomplete or nonspecific, can alter the target's interactions with other molecules, provides only moderate precision of localization and

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Section: Methodsmentioning

confidence: 94%

Label-free single-instance protein detection in vitrified cells

Choi

Lippincott‐Schwartz

et al. 2020

Preprint

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“…Ice thickness can vary depending on the dimensions of the particle but usually ranges from a few nanometres to a hundred nanometres [48]. The thicker the ice (>100 nm), the worse the resolution [49]. 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 [47].…”

Section: Filtrationmentioning

confidence: 99%

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

et al. 2020

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Sterilisation and preservation of vesicle formulations are important considerations 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-block-poly(ethylene oxide) polymers (PBd 12 -PEO 11 and PBd 22 -PEO 14 ) mixed with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (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 the 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.Polymers 2020, 12, 914 2 of 17 liposomes can have inherent instabilities. Their membranes are highly flexible under bending deformations, but weak under stretching deformations, with a lysis strain of less than 5% [28,29]. The labile fluidity of the membrane can lead to transient membrane defects that frustrates long term encapsulation stability and lipid peroxidation can cause chemical instabilities in these structures.Polymersomes, due to their synthetic nature, are often less biocompatible than their lipid counterparts but offer greater mechanical stability and a broader chemical parameter space [30]. Amphiphilic copolymers that form vesicles can have several different architectures, the most common being AB diblock (A = hydrophilic, B = hydrophobic) [6], ABA [31] and ABC tri-block polymers [32] where A and C are chemically different hydrophilic blocks and finally graft copolymers [33]. Formation of polymersomes depends on the amphiphilic co-polymers molecular weight, polydispersity and hydrophilic/hydrophobic block lengths ratio, which is ideally less than 1:3 to form vesicular structures [34]. Their membranes are often thicker than liposome membranes, which provides greater bending resistance and their enhanced elasticity under stretching makes them tough and durable [22]. The polymer chemistry can be designed to minimise chemical instability but also to incorporate novel functionality. In blending liposomes and polymersomes to create hybrid vesicles, the ambition is to combine advantageous properties of these m...

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“…Ice thickness can vary depending on the dimensions of the particle but usually range from a few nanometres to a hundred nanometres [44]. 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.

show abstract

Routine determination of ice thickness for cryo-EM grids

Cited by 130 publications

References 37 publications

Label-free single-instance protein detection in vitrified cells

Label-free single-instance protein detection in vitrified cells

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

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

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