Spotiton: New features and applications

Dandey, Venkata P.; Wei, Hui; Zhang, Zhening; Tan, Yong Zi; Acharya, Priyamvada; Eng, Edward T.; Rice, William J.; Kahn, Peter; Potter, Clinton S.; Carragher, Bridget

doi:10.1016/j.jsb.2018.01.002

Cited by 144 publications

(111 citation statements)

References 22 publications

(24 reference statements)

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“…Grid reproducibility and obtaining suitable ice thickness were issues encountered when using manual grid preparation. To address these issues, the samples were vitrified using a semi-automated Spotiton V1.0 robot 15,16,36 , a novel device for preparing cryoEM samples that uses piezo-electric dispensing to apply small (50pL) drops of sample across a “self-blotting” nanowire grid as it flies past en route to plunge into liquid ethane. The nanowire grids, manufactured in house, used either lacey carbon or lacey gold supports 37 ; the gold support helps to optimize image quality when acquiring data with the grid tilted relative to the electron beam 14,38 .…”

Section: Methodsmentioning

confidence: 99%

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Scapin

Dandey

Zhang

et al. 2018

Nature

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Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

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Section: Methodsmentioning

confidence: 99%

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Scapin

Dandey

Zhang

et al. 2018

Nature

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192

236

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“…The Spotiton system 18, 19 was upgraded with a second set of identical dispense head components. A second piezo driven electric tip was mounted next to the first head 4.5 mm from the first tip (Figure S1b).…”

Section: Methodsmentioning

confidence: 99%

“…We have developed a new approach to the spraying-mixing method based on the Spotiton robot 18, 19 that uses a piezo dispensing tip to apply a stream of ~50 pL droplets onto a nanowire (“self-wicking”) grid 20 as it rapidly speeds past on its way to vitrification in a liquid cryogen. This method produces a stripe of ice of fairly uniform thickness across each grid, which is often sufficient to acquire enough data for a high-resolution map.…”

Section: Figurementioning

confidence: 99%

Time-resolved cryoEM using Spotiton

Dandey

Wei

Bobe

et al. 2020

Preprint

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28We present an approach for preparing cryoEM grids to study short-lived molecular states. 29Using piezo electric dispensing, two independent streams of ~50 pL sample drops are 30 deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing 31 reaction stops when the grid is vitrified in liquid ethane, on the order of ~100 ms later. We 32 demonstrate the utility of this approach for four biological systems where short-lived 33states are of high interest. 34 38 Chen, J. et al. E. coli TraR allosterically regulates transcription initiation by altering RNA 541 polymerase conformation. eLife 8, doi:10.7554/eLife.49375 (2019). 542 39 Posson, D. J., Rusinova, R., Andersen, O. S. & Nimigean, C. M. Calcium ions open a 543 selectivity filter gate during activation of the MthK potassium channel. Nat Commun 6, 544 8342,

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“…Several methods have been presented to improve sample preparation for cryo-EM, all having their own speci c purpose. Examples include high-throughput grid preparation [25][26][27], time-resolved EM [28], and single-cell visual proteomics [11,12]. Furthermore, antibody-functionalized EM-grids were proposed to ' sh' target proteins [29,30], and a nity grids designed to capture proteins with engineered his-tags [31].…”

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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Spotiton: New features and applications

Cited by 144 publications

References 22 publications

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Time-resolved cryoEM using Spotiton

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

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