Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, the use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes.

Mass spectrometers as cryoEM grid preparation instruments.

Structure determination by single-particle cryoEM has matured into a core structural biology technique. Despite many methodological advancements, most cryoEM grids are still prepared using the plunge-freezing method developed ∼40 years ago. Embedding samples in thin films and exposing them to the air-water interface often leads to sample damage and preferential orientation of the particles.

Cryogenic Soft Landing Improves Structural Preservation of Protein Complexes.

We describe an apparatus for the cryogenic landing of particles from the ion beam of a mass spectrometer onto transmission electron microscope grids for cryo-electron microscopy. This system also allows for the controlled formation of thin films of amorphous ice on the grid surface. We demonstrate that as compared to room temperature landings, use of this cryogenic landing device greatly improves the structural preservation of deposited protein-protein complexes.

Onto Grid Purification and 3D Reconstruction of Protein Complexes Using Matrix-Landing Native Mass Spectrometry.

Addressing mixtures and heterogeneity in structural biology requires approaches that can differentiate and separate structures based on mass and conformation. Mass spectrometry (MS) provides tools for measuring and isolating gas-phase ions. The development of native MS including electrospray ionization allowed for manipulation and analysis of intact noncovalent biomolecules as ions in the gas phase, leading to detailed measurements of structural heterogeneity.

Matrix-Landing Mass Spectrometry for Electron Microscopy Imaging of Native Protein Complexes.

Recently, we described the use of a chemical matrix for landing and preserving the cations of protein-protein complexes within a mass spectrometer (MS) instrument. By use of a glycerol-landing matrix, we used negative stain transmission electron microscopy (TEM) to obtain a three-dimensional (3D) reconstruction of landed GroEL complexes. Here, we investigate the utilities of other chemical matrices for their abilities to land, preserve, and allow for direct imaging of these cationic particles using TEM.

Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles and have linked their dysfunction to more than 150 distinct disorders. Still, hundreds of mitochondrial proteins lack clear functions, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved.

Three-dimensional structure determination of protein complexes using matrix-landing mass spectrometry.

Native mass spectrometry (MS) is increasingly used to provide complementary data to electron microscopy (EM) for protein structure characterization. Beyond the ability to provide mass measurements of gas-phase biomolecular ions, MS instruments offer the ability to purify, select, and precisely control the spatial location of these ions. Here we present a modified Orbitrap MS system capable of depositing a native MS ion beam onto EM grids.

Selective binding of anions by rigidified nanojars: sulfate carbonate.

Selective binding and transport of highly hydrophilic anions is ubiquitous in nature, as anion binding proteins can differentiate between similar anions with over a million-fold efficiency. While comparable selectivity has occasionally been achieved for certain anions using small, artificial receptors, the selective binding of certain anions, such as sulfate in the presence of carbonate, remains a very challenging task. Nanojars of the formula [anion⊂{Cu(OH)(pz)}] (pz = pyrazolate; = 27-33) are totally selective for either CO or SO over anions such as NO, ClO, BF, Cl, Br and I, but cannot differentiate between the two.