Addressing preferred specimen orientation in single-particle cryo-EM through tilting

Tan, Yong Zi; Baldwin, Philip R.; Davis, Joseph H.; Williamson, James R.; Potter, Clinton S; Carragher, Bridget; Lyumkis, Dmitry

doi:10.1038/nmeth.4347

Cited by 771 publications

(756 citation statements)

References 52 publications

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“…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 . Tilted data collection was necessary to address highly preferred particle orientation observed during initial data collections.…”

Section: Methodsmentioning

confidence: 99%

“…The defocus ranged from −1 to −2.5 μm. Images were collected at a tilt angle of −30° to address the issue of preferred orientation identified during initial data collection 14 ; exposure was set to 10 sec (40 frames/image), for a total dose of ~68 e − /Å 2 . A total of 6805 images were collected in three sessions using Leginon 40 .…”

Section: Methodsmentioning

confidence: 99%

“…The resolution of each reconstruction was determined by the gold standard Fourier shell correlation criterion in Cryosparc 43 and Relion PostProcess 44 . The completeness of the maps was assessed by measuring the Euler angle orientation distribution (as calculated by Cryosparc 43 ) and by evaluating each map 3D FSC 14 . (Extended Data Figures 2, 3, and 6).…”

Section: Methodsmentioning

confidence: 99%

“…c , Gold standard FSC curve as calculated in RELION 44 . d , Plot of the global half-map FSC (solid red line) and spread of directional resolution values (+/− 1σ from mean, green dotted lines; the blue bars are a histogram of 100 such values evenly sampled over the 3D FSC 14 . e , Local resolution distribution (as calculated in Cryosparc 43 ).…”

Section: Extended Datamentioning

confidence: 99%

“…A pronounced preferred orientation of the complex in vitreous ice was overcome using a combination of tilted data collection 14 and fast plunge speeds using the Spotiton instrument 15,16 . Further details are provided in the methods section.…”

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confidence: 99%

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

Scapin

Dandey

Zhang

et al. 2018

Nature

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193

243

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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%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Extended Datamentioning

confidence: 99%

mentioning

confidence: 99%

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

Scapin

Dandey

Zhang

et al. 2018

Nature

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193

243

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Cryo‐Electron Microscopy in Drug Discovery and Development: Opportunities and Challenges

Gomez‐Llorente

Hong

Scapin

2021

Burger's Medicinal Chemistry and Drug Discovery

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While utilization of structural information has been a staple technology in drug discovery for many years, electron microscopy creates opportunities that have been thus far out of reach for the conventional techniques, like the visualization of very large molecular complexes in solution or obtaining high‐resolution structural data for small molecules from microcrystals. These opportunities come at a cost: hardware is extremely expensive, software is ever‐changing, and constantly challenged by new developments in the hardware and requests by the users, and IT departments are faced with significant requirements for storage and computational power. Nevertheless, the establishment of centralized facilities, commercial consortia, and cooperative research centers is well positioned to provide access to Cryo‐EM to an expanded base of researchers, from academia as well as from industry. Understanding of the requirements, advantages, and limitations of this technique becomes then necessary to ensure correct decision‐making processes and resource allocation when undertaking a structure‐guided drug design project.

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Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

Januliene

Moeller

2020

FEBS Letters

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High-resolution cryo-EM has revolutionized how we look at ABC transporters and membrane proteins in general. An ever-increasing number of software tools and faster processing now allow dissecting the molecular details of nanomachines at atomic precision. Considering the further benefits of significantly reduced sample demands and increased speed, cryo-EM will dominate the structure determination of membrane proteins in the near future without compromising on data quality or detail. Moreover, improved and new algorithms make it now possible to resolve the conformational spectrum of macromolecular machines under turnover conditions and to analyze heterogeneous samples at high-resolution. The future of cryo-EM is, therefore, bright, and the growing number of imaging facilities and groups active in this field will amplify this trend even further. Nevertheless, expectations have to be managed, as cryo-EM alone cannot provide an ultimate answer to all scientific questions. In this review, we discuss the capabilities and limitations of cryo-EM together with possible solutions for studies of ABC transporters.

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Addressing preferred specimen orientation in single-particle cryo-EM through tilting

Cited by 771 publications

References 52 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

Cryo‐Electron Microscopy in Drug Discovery and Development: Opportunities and Challenges

Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

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