Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy

Wang, Feng; Yu, Zanlin; Betegon, Miguel; Campbell, Melody G.; Aksel, Tural; Zhao, Jianhua; Li, Sam; Douglas, Shawn M.; Agard, David A.

doi:10.1016/j.jsb.2019.107437

Cited by 48 publications

(44 citation statements)

References 12 publications

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“…For high-resolution cryo-EM, the apo-FPN-Fab45D8 complex was diluted to 0.0375 mg/mL in 20 mM HEPES pH 7.5, 100 mM NaCl directly prior to vitrification, and 2 μL sample was applied to glow-discharged gold holey carbon 1.2/1.3 300-mesh grids (Quantifoil) coated in-house with graphene oxide 51 – 53 . Grids were blotted for 2–4 seconds at 0 force and 10 seconds wait time before being plunge vitrified in liquid ethane using a MarkIV Vitrobot (ThermoFisher).…”

Section: Methodsmentioning

confidence: 99%

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms

Billesboelle

Azumaya

Kretsch

et al. 2020

Nature

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217

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The serum iron level in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing ferroportin internalization and degradation 1 . Aberrant ferroportin activity can lead to diseases of iron overload, like hemochromatosis, or iron limitation anemias 2 . Here, we determined cryogenic electron microscopy (cryo-EM) structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with cobalt, an iron mimetic, and hepcidin. These structures and accompanying molecular dynamics simulations identify two metal binding sites within the N- and C-domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy-terminus of hepcidin directly contacts the divalent metal in the ferroportin C-domain. We further show that hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, where only iron loaded ferroportin molecules are targeted for degradation. More broadly, our structural and functional insights are likely to enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

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Section: Methodsmentioning

confidence: 99%

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms

Billesboelle

Azumaya

Kretsch

et al. 2020

Nature

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217

186

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“…Assuming 10.5 bp per helical turn, a 1-nt shift corresponds to a 3.4 Å translation along the stage axis, and a 34.3° rotation. To modify the stage rotation angle, we can modify the scaffold route in the surrounding anchoring region (dark gray, A pooled mixture with equal fractions of all goniometer designs are adsorbed onto holey gold grids with an amino-graphene oxide support 15 ; Fig. 1H shows a representative micrograph region.…”

Section: Resultsmentioning

confidence: 99%

“…Prior to sample deposition and negative staining, ultrathin carbon coated quantifoil grids (Pacific grid tech, CA) were treated with a Pelco glow discharge unit (30 seconds hold/30 seconds glow discharge). For negative-stain grid preparation, 5 µl of 80 ng/ul DNA origami goniometer sample was deposited onto a glow-discharged ultra thin-carbon coated, graphene-oxide (made in-house) or amine-functionalized graphene-oxide grids (made in-house as described 15 ). The sample was incubated on the grids for 1 minute.…”

Section: Negative Stain Tem Experimentsmentioning

confidence: 99%

Molecular goniometers for single-particle cryo-EM of DNA-binding proteins

Aksel

Cheng

et al. 2020

Preprint

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Correct reconstruction of macromolecular structure by cryo-electron microscopy relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. DNA nanotechnology was conceived as a potential tool for building host nanostructures to prescribe the locations and orientations of docked proteins.We used DNA origami to construct molecular goniometers-instruments to precisely orient objects-to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. Each protein orientation maps to a distinct barcode pattern specifying particle classification and angle assignment. We used goniometers to obtain a 6.5 Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using classical cryo-EM. Our approach should be adaptable for other DNA-binding proteins, and a wide variety of other small proteins, by fusing DNA binding domains to them.

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“…(b) Graphene oxide is an oxygenated, hydrophilic form of graphene, in which pristine areas are interspersed with oxidized carbon (Mkhoyan et al ., 2009). Oxidized graphene can be chemically modified to generate functionalized surfaces for specific protein attachment (Wang et al ., 2020 a , 2020 b ). Graphene oxide is typically sold as a colloidal suspension of – usually multi-layered – flakes (Bokori-Brown et al ., 2016; Boland et al ., 2017; Coscia et al ., 2020).…”

Section: Continuous Support Filmsmentioning

confidence: 99%

Current limitations to high-resolution structure determination by single-particle cryoEM

D’Imprima

Kühlbrandt

2021

Quart. Rev. Biophys.

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CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam-induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. We examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution.

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Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy

Cited by 48 publications

References 12 publications

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms

Molecular goniometers for single-particle cryo-EM of DNA-binding proteins

Current limitations to high-resolution structure determination by single-particle cryoEM

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