Cryo-EM sample preparation method for extremely low concentration liposomes

Tonggu, Lige; Wang, Liguo

doi:10.1101/494997

Cited by 9 publications

(13 citation statements)

References 51 publications

(54 reference statements)

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“…For single-component membranes, it is generally assumed that the liposome population obtained from freeze-thaw/extrusion generates a uniform population with diameters matching the size of the extrusion pore (Figure 2A). Indeed, cryo-electron microscopy studies of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) liposomes extruded through 50 nm pores show a distribution with a mean of 27 nm (23). In addition, egg PC vesicles extruded through 400 nm pores is reported to be “relatively homogeneous” with a distribution centered at 243 ± 91 nm (Figure 2B), as measured by freeze-fracture electron microscopy (12).…”

Section: Resultsmentioning

confidence: 99%

Occupancy distributions of membrane proteins in heterogeneous liposome populations

Cliff

Chadda

Robertson

2019

Preprint

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11Measurements of membrane protein structure and function often rely on reconstituting the protein 12 into lipid bilayers through the formation of liposomes. Many measurements conducted in 13 proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, 14 require some understanding of the occupancy statistics of the liposome population for correct 15 interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein 16 incorporation can be described by the Poisson distribution. However, in reality, liposomes are 17 heterogeneous, which alters the statistics of occupancy in several ways. Here, we determine the 18 liposome occupancy distribution for membrane protein reconstitution while taking into account 19 liposome size heterogeneity. We calculate the protein occupancy for a homogenous population of 20 liposomes with radius r = 200 nm, representing an idealization of vesicles extruded through 400 21 nm pores and compare it to the right-skewed distribution of 400 nm 2:1 POPE:POPG vesicles. As 22 than one protein at higher protein/lipid densities. These results demonstrate that the occupancy 32 distributions of membrane proteins into vesicles can be accurately predicted in heterogeneous 33 populations with experimental knowledge of the liposome size distribution. 34 35 Keywords 36 Liposome size distribution, Poisson, membrane protein, reconstitution, single-molecule 37 photobleaching, CLC-ec1 38 39 Abbreviations 40micelle concentration. From this mixed-micelle condition, the detergent is then slowly removed 63 by dialysis, resulting in the formation of bilayers with the protein incorporated within, that then 64 assemble into self-contained liposomes (4). 65 66One of the benefits of the compartmentalized structure of liposomes is that it enables the 67 establishment of gradients across the membrane, which allows interrogation of the protein's 68 function (5, 6). One limitation, is that dialysis methods typically generate small, unilamellar 69 vesicles (7) that incorporate the protein according to the state in the mixed micelle condition. Since 70 liposomes do not spontaneously exchange with one another on a reasonable time scale, this means 71 that the protein state that is examined in the resultant proteoliposome population may not reflect 72 equilibrium. This presents an issue for considering stoichiometry of the protein, particularly at 73 'Poisson-dilutions' where most of the liposomes are unoccupied or have a single protein species. 74However, this is the range of dilution that is most desirable when considering protein function, as 75 the membrane transport behavior reflects a single-molecule and so unitary transport rates can be 76 estimated (8). Still, when investigating the function of the native assembly, it is advantageous to 77 capture the equilibrium state of the protein in the membrane into single vesicles for further analysis 78 of function and dynamics. 79 80One way of doing this is by taking the small unilamellar liposomes from ...

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Section: Resultsmentioning

confidence: 99%

Occupancy distributions of membrane proteins in heterogeneous liposome populations

Cliff

Chadda

Robertson

2019

Preprint

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“…In addition, we reconstitute NaChBac into liposomes for structural investigation, which can provide more orientations of the protein for single-particle analysis, as well as reproducing more of the proteins' native physiological environments. The long-time historical challenge to investigate the structure of lipoproteins is the low density of liposomes on a cryo-EM holey carbon grids (27,28) (Fig. 5C).…”

Section: High Density Of Membrane and Lipoproteins On Graphene Gridsmentioning

confidence: 99%

High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy

Han

Fan

Wang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

123

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Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids—aimed at preserving thin, uniform vitrified ice and improving protein adsorption—have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with up to 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6-Å resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipoproteins by at least 5-fold, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, this approach supports customized grid designs targeting specific proteins, owing to its broad compatibility with a variety of nanomaterials.

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“…More recently, imaging liposome-embedded MPs using SP cryo-EM has been developed based on the method of random spherically constrained SP reconstruction. This approach relies on fitting and subtracting a model of the membrane contribution to each image and reconstituting the MP particles alone (Wang and Sigworth, 2010; Tonggu and Wang, 2020). It has yielded cryo-EM structures of the large conductance voltage- and calcium-activated potassium (BK) channels first at 17 Å (Wang and Sigworth, 2009), and then at 3.5 Å (Tonggu and Wang, 2018).…”

Section: An Overview Of Surfactant Usage At the Vitrification Stepmentioning

confidence: 99%

Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy

Bon

Michon

Popot

et al. 2021

Quart. Rev. Biophys.

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Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.

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Cryo-EM sample preparation method for extremely low concentration liposomes

Cited by 9 publications

References 51 publications

Occupancy distributions of membrane proteins in heterogeneous liposome populations

Occupancy distributions of membrane proteins in heterogeneous liposome populations

High-yield monolayer graphene grids for near-atomic resolution cryoelectron microscopy

Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy

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