Mercury-containing nucleotides for phasing applications with nucleotide- or DNA-binding proteins

Selenium-containing nucleotides for phasing applications with nucleotide- or DNA-binding proteins

Single particle electron cryo-microscopy (cryo-EM) is a powerful technique to determine the structures of protein complexes down to atomic resolution. The sample in solution is flash-frozen in liquid ethane, forming a thin layer of vitreous ice, in which the sample particles are ideally evenly distributed in random orientation far enough from the air-water interface (pictured below).
However, protein sample vitrification for cryo-EM is a major bottleneck. Only the combination of choosing the right grid type, vitrification conditions, protein concentration and additives enables high quality data acquisition in cryo-EM.
The Cryo-EM V-Kit is a Vitrification Starter Kit that offers both a selection of surfactants which have been successfully applied in cryo-EM sample preparation and a selection of Quantifoil Holey Carbon Films to facilitate the search for the optimal vitrification condition for both soluble and membrane proteins.

Schematic cross section of a grid hole with ideally distributed single particles in random orientation

Quantifoil® is a holey carbon film with a thickness of about 12 nm, that is applied on a standard copper or gold electron microscopy grid and provides an ideal support for biological samples in cryo-EM techniques^[1-8].
The supporting mesh (copper or gold) is available with distinct numbers. Higher mesh numbers (400) indicate closely spaced bars and provide higher stability, whereas lower numbers (200) provide larger free faces.
The Quantifoil® carbon film has circular holes of defined size and interspace, e.g. R 2/1 (pictured) has holes of 2 µm diameter with an interspace of 1 µm in both dimensions. Higher magnifications usually require smaller holes and vice versa.

You have the following options to choose from:

Quantifoil® film

• R 0.6/1 (0.6 µm hole diameter/1 µm interspace)
• R 1.2/1.3 (1.2 µm hole diameter/1.3 µm interspace)
• R 2/1 (2 µm hole diameter/1 µm interspace)
• R 2/2 (2 µm hole diameter/2 µm interspace)
• R 3.5/1 (3.5 µm hole diameter/1 µm interspace)

Supporting mesh material

• Copper
• Gold

Supporting mesh number

• 200
• 300
• 400

Selected Recent Literature Citations of Quantifoil®

[1] Lee et al. (2019) Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation. Cell Reports 26:29.
[2] Azubel et al. (2019) FGF21 trafficking in intact human cells revealed by cryo-electron tomography with gold nanoparticles. eLife DOI:10.7554/eLife.43146.
[3] Zhao et al. (2018) Structure and mechanogating mechanism of the Piezo1 channel. Nature 554:487.
[4] Wijnands et al. (2018) Controlling protein activity by dynamic recruitmenton a supramolecular polymer platform. Nat. Commun. 9:65.
[5] Ke et al. (2018) Promotion of virus assembly and organization by the measles virus matrix protein. Nat. Commun. 9:1736.
[6] Zhang et al. (2018) Molecular structure of the ATP-bound, phosphorylated human CFTR. PNAS 115:12757.
[7] Park et al. (2017) Structure of a CLC chloride ion channel by cryo-electron microscopy. Nature 541:500.
[8] Ekiert et al. (2017) Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 169:273.

Quantifoil® is a holey carbon film with a special geometry and a thickness of about 12 nm, that is applied on a standard copper electron microscopy grid and provides an ideal support for biological samples in cryo-EM techniques^[1-8]. Higher mesh numbers (300) indicate closely spaced bars and provide higher stability, whereas lower numbers (200) provide larger free faces. The Quantifoil® carbon film has circular holes of defined size and interspace, e.g. R 2/1 (pictured) has holes of 2 µm diameter with an interspace of 1 µm in both dimensions.
The C2 versions on this page provide an additional 2 nm continuous carbon layer on top.

Selected Recent Literature Citations of Quantifoil®

[1] Lee et al. (2019) Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation. Cell Reports 26:29.
[2] Azubel et al. (2019) FGF21 trafficking in intact human cells revealed by cryo-electron tomography with gold nanoparticles. eLife DOI:10.7554/eLife.43146.
[3] Zhao et al. (2018) Structure and mechanogating mechanism of the Piezo1 channel. Nature 554:487.
[4] Wijnands et al. (2018) Controlling protein activity by dynamic recruitmenton a supramolecular polymer platform. Nat. Commun. 9:65.
[5] Ke et al. (2018) Promotion of virus assembly and organization by the measles virus matrix protein. Nat. Commun. 9:1736.
[6] Zhang et al. (2018) Molecular structure of the ATP-bound, phosphorylated human CFTR. PNAS 115:12757.
[7] Park et al. (2017) Structure of a CLC chloride ion channel by cryo-electron microscopy. Nature 541:500.
[8] Ekiert et al. (2017) Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 169:273.

UltrAuFoil® is a holey gold film with a thickness of about 500 Å, that provides an ultrastable gold support for for biological samples in cryo-EM techniques. The movement of frozen specimens during imaging will be significantly reduced leading to better image contrast and finally to better 3D reconstructions with less data^[1].

Reference

[1] Russo et al. (2014) Ultrastable gold substrates for electron cryomicroscopy. Science 346:1377.

The Carbon Support Film is a continuous carbon film with a thickness of 10 nm, typically used for negative staining of biological samples in cryo-em techniques.
Quantifoil Multi Holes offer a pattern of various hole sizes, shapes and arrangements and are typically used in cryo-tomography (cryo-ET) or cryo-electron microscopy (cryo-EM) techniques.
Lacey Carbon Films offer a lacey mesh structure with a high percentage of open area. The hole sizes vary significantly and the bars are extremely thin.

C-flat™ is an ultra-flat, holey carbon film with a thickness of about 20 nm supported by a standard transmission electron microscopy (TEM) grid. The precise patent pending technology by which C-flat™ is manufactured eliminates artifacts such as excess carbon and edges around holes. The ultra-flat surface results in better particle dispersion and more uniform ice thickness. Manufactured without plastics, C-flat™ grids are clean upon arrival and the user has no residue to contend with. The C-flat™ carbon film has circular holes of defined size and interspace, e.g. CF 2/2 (pictured) has holes of 2 µm diameter with an interspace of 2 µm in both dimensions.

You have the following options to choose from:

C-flat™ film

• CF 1.2/1.3 (1.2 µm hole diameter/1.3 µm hole spacing)
• CF 2/1 (2 µm hole diameter/1 µm hole spacing)
• CF 2/2 (2 µm hole diameter/2 µm hole spacing)

Supporting TEM grid material

• Copper
• Gold

Supporting TEM grid mesh size

• 200
• 300
• 400

Looking for gold? Check out the Au-flat™ Holey Gold Films!

Au-flat™ is an ultrastable holey gold alloy film (80% Au and 20% Pd) with a thickness of about 45 nm supported by a transmission electron microscopy (TEM) gold grid. It’s a derivative of the patented C-flat™ holey carbon films.

• significantly reduces beam-induced motion during imaging compared to carbon films, improving image quality and resolution
• significantly stronger than carbon films and more capable of surviving the cryo-EM workflow including tweezer handling, glow discharge, auto-grid loading and plunge freezing
• chemically inert and bio-compatible

Fast delivery – most Au-flat™ grids listed below are currently in stock

Amphipols are short amphipatic polymers that are specifically designed to stabilize membrane proteins in aqueous solutions.
Due to their dense distribution of hydrophobic chains they tightly bind to transmembrane surfaces of membrane proteins and cover it with a thin interfacial layer of surfactant^[1]. The resulting small hydrophilic complexes have several advantages over usually much larger protein detergent complexes, such as stability and functionality of the membrane protein.
Amphipol A8-35 is successfully applied as stabilizing agent in Cryo-EM^[2-5] and X-ray crystallography^[6].

References

[1] Zoonens et al. (2014) Amphipols for Each Season. J Membrane Biol 247:759.
[2] Chen et al. (2016) Structure of the STRA6 receptor for retinol uptake. Science 353:887.
[3] Zubcevic et al. (2016) Cryo-Electron Microscopy of the Trpv2 Ion Channel. Nat Struct Mol Biol 23:180.
[4] Bai et al. (2015) Sampling the conformational space of the catalytic subunit of human gamma-secretase. DOI 10.7554/eLife.11182.
[5] Althoff et al. (2011) Arrangement of electron transport chain components in bovine mitochondrial supercomplex I₁III₂IV₁. EMBO J 30:4652.
[6] Polovinkin et al. (2014) High-Resolution Structure of a Membrane Protein Transferred from Amphipol to a Lipidic Mesophase. J Membrane Biol 247:997.