A new method for vitrifying samples for cryoEM

Razinkov, Ivan; Dandey, Venkata P.; Wei, Hui; Zhang, Zhening; Melnekoff, David; Rice, William J.; Wigge, Christoph; Potter, Clinton S.; Carragher, Bridget

doi:10.1016/j.jsb.2016.06.001

Cited by 137 publications

(133 citation statements)

References 19 publications

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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%

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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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192

236

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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%

“…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.…”

mentioning

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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“…Furthermore, competitors are trying to redress the imbalance with new and improved solutions; for instance, Jeol with a new automatic specimen-loading system, a redesigned FREALIGN with a graphical user interface (cisTEM; personal communication with Timothy Grant, Janelia Research Campus), and the FEI Falcon III camera, which will even be retrofitted onto the most recently sold FEI microscope systems with a Falcon II camera. Finally, there are a number of nascent technological developments such as phase plates (Danev & Baumeister, 2016) and specimen-preparation robots [for example Spotiton (Razinkov et al, 2016) from the Carragher laboratory at NYSBC and Vitrojet from the Peters laboratory at Maastricht University] that may reach a level of maturity in the next few years to significantly impact the cryo-EM field and depositions to EMDB. It may therefore be useful to repeat the analysis of EMDB trends on an annual or biannual basis to factor in such developments.…”

Section: Discussionmentioning

confidence: 99%

Trends in the Electron Microscopy Data Bank (EMDB)

Patwardhan

2017

Acta Cryst Sect D Struct Biol

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Recent technological advances, such as the introduction of the direct electron detector, have transformed the field of cryo-EM and the landscape of molecular and cellular structural biology. This study analyses these trends from the vantage point of the Electron Microscopy Data Bank (EMDB), the public archive for three-dimensional EM reconstructions. Over 1000 entries were released in 2016, representing almost a quarter of the total number of entries (4431). Structures at better than 6 Å resolution now represent one of the fastest-growing categories, while the share of annually released tomography-related structures is approaching 20%. The use of direct electron detectors is growing very rapidly: they were used for 70% of the structures released in 2016, in contrast to none before 2011. Microscopes from FEI have an overwhelming lead in terms of usage, and the use of the RELION software package continues to grow rapidly after having attained a leading position in the field. China is rapidly emerging as a major player in the field, supplementing the US, Germany and the UK as the big four. Similarly, Tsinghua University ranks only second to the MRC Laboratory for Molecular Biology in terms of involvement in publications associated with cryo-EM structures at better than 4 Å resolution. Overall, the numbers point to a rapid democratization of the field, with more countries and institutes becoming involved.

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“…A more radical approach (“Spotiton”) that potentially eliminates the need for the process of blotting prior to plunge-freezing has been proposed [24, 70]. In this procedure, picoliter sized amounts of protein solution are delivered to individual grid squares, and can be used in conjunction with grids specially modified with carbon nanowires to facilitate better spreading of the aqueous film by capillary action [71]. …”

Section: Grid Modification Methods For Specimen Preparationmentioning

confidence: 99%

Cryo-EM: beyond the microscope

Earl

Falconieri

Milne

et al. 2017

Current Opinion in Structural Biology

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The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination, and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.

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A new method for vitrifying samples for cryoEM

Cited by 137 publications

References 19 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

Trends in the Electron Microscopy Data Bank (EMDB)

Cryo-EM: beyond the microscope

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