Single-cell lysis for visual analysis by electron microscopy

Kemmerling, Simon; Arnold, Stefan; Bircher, Benjamin A.; Sauter, Nora; Escobedo, Carlos; Dernick, Gregor; Hierlemann, Andreas; Stahlberg, Henning; Braun, Thomas

doi:10.1016/j.jsb.2013.06.012

Cited by 33 publications

(48 citation statements)

References 38 publications

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“…The use of a more controllable specimen dispensing system (e.g. Spotiton [47] and micro-capillary method [48]) may also help minimizing the sample consumption.…”

Section: Time-resolved Cryo-emmentioning

confidence: 99%

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

Chen

Frank²

2015

Microscopy (Tokyo)

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The capabilities and application range of cryogenic electron microscopy (cryo-EM) method have expanded vastly in the last two years, thanks to the advances provided by direct detection devices and computational classification tools. We take this review as an opportunity to sketch out promising developments of cryo-EM in two important directions: (i) imaging of short-lived states (10-1000 ms) of biological molecules by using timeresolved cryo-EM, particularly the mixing-spraying method and (ii) recovering an entire continuum of coexisting states from the same sample by employing a computational technique called manifold embedding. It is tempting to think of combining these two methods, to elucidate the way the states of a molecular machine such as the ribosome branch and unfold. This idea awaits further developments of both methods, particularly by increasing the data yield of the time-resolved cryo-EM method and by developing the manifold embedding technique into a user-friendly workbench.

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“…The use of a more controllable specimen dispensing system (e.g. Spotiton [47] and micro-capillary method [48]) may also help minimizing the sample consumption.…”

Section: Time-resolved Cryo-emmentioning

confidence: 99%

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

Chen

Frank²

2015

Microscopy (Tokyo)

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“…The use of openBEB together with our recent hardware developments that connect micro-fluidics to EM for visual proteomics [8,10] is reported. In the demonstration experiment adherent eukaryotic cells are cultured, and monitored by time-lapse LM throughout.…”

Section: Resultsmentioning

confidence: 99%

“…In the demonstration experiment adherent eukaryotic cells are cultured, and monitored by time-lapse LM throughout. At specific time points, pulse chase investigations are performed and, after a chase time, individual cells are lysed by electroporation [10]. Subsequently, the cell lysate is transferred into a microcapillary, prepared for EM and imaged for analysis by “lyse and spread visual proteomics” [8,9].…”

Section: Resultsmentioning

confidence: 99%

openBEB: open biological experiment browser for correlative measurements

et al. 2014

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BackgroundNew experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis.ResultsWe have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository.ConclusionsThe versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the “house keeping” abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.

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“…In addition, further optimization of the puri cation step and use of the cryoWriter, will almost certainly make it possible to perform the whole preparation in less than 1 h, facilitating the investigation of sensitive proteins targets. Finally, the toolchain presented here can also be combined with in vitro translation systems, which would enable high-throughput structure determination, or with a single cell lysis device [11,12,32], which would bring the structural analysis of proteins originating from a single cell within reach.…”

Section: Microcapillarymentioning

confidence: 99%

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

Schmidli

Albiez

Rima

et al. 2019

Preprint

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High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousands to a few million protein particles required for structure-determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a micro uidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 µL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens new opportunities for the isolation of sensitive, endogenous protein complexes.Knowledge of a protein's architecture at high resolution is vital to understand its mechanics and chemistry. In recent years, cryogenic electron microscopy (cryo-EM) [1] has matured into a powerful method that can determine the architecture of biological macromolecules at 1 the resolutions required to interpret the atomic fold of proteins [2, 3]. In the single-particle cryo-EM approach[4], an unsupported, thin layer of isolated protein complexes in amorphous (vitri ed) ice is visualized at close to physiological conditions. Only several thousand to a few million imaged particles are needed to calculate a high-resolution, three-dimensional (3D) structure. Nevertheless, protein production, puri cation, and sample preparation for cryo-EM are nowadays considered the bottleneck for structure determination [5][6][7]. We have identi ed two dominating reasons for this: Firstly, signi cant amounts of protein must be produced. Conventional sample preparation for cryo-EM requires several microliters of a puri ed protein solution at a concentration of approx. 1 mg∕mL per grid, from which extensive lter-paper blotting later removes the vast majority of protein particles [8, 9]. Secondly, both, protein puri cation and cryo-EM sample preparation are lengthy and harsh procedures.Mostly, high-yield expression systems are employed, and one or two chromatographic steps are needed to purify the protein particles. In addition, the classical cryo-EM sample preparation process that follows is a rough procedure [10], primarily because of the blotting step, and many proteins denature.We recently developed a micro uidic cryo-EM grid preparation system termed cryoWriter, allowing the preparation of cryo-EM specimens from nanoliters of sample-solution [11][12][13].Since the cryoWriter does not use paper blotting, it ensures that grid preparation is gentle and virtually lossless. Here, we report the combination of sample grid preparation using the cryoWriter with micro uidic protein puri cation [14], to determine the 3.5 Å cryo-EM structure of the untagged human 20S proteasome complex, which is the 'catalytic core' of the ubiquitin-proteasome system involved i...

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Single-cell lysis for visual analysis by electron microscopy

Cited by 33 publications

References 38 publications

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

openBEB: open biological experiment browser for correlative measurements

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

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