Cryo-EM of soft-landed ß-galactosidase: Gas-phase and native structures are remarkably similar.

Native mass spectrometry (MS) has become widely accepted in structural biology, providing information on stoichiometry, interactions, homogeneity, and shape of protein complexes. Yet, the fundamental assumption that proteins inside the mass spectrometer retain a structure faithful to native proteins in solution remains a matter of intense debate. Here, we reveal the gas-phase structure of ß-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.6-Å resolution, enabled by soft landing of mass-selected protein complexes onto cold transmission electron microscopy (TEM) grids followed by in situ ice coating. We find that large parts of the secondary and tertiary structure are retained from the solution. Dehydration-driven subunit reorientation leads to consistent compaction in the gas phase. By providing a direct link between high-resolution imaging and the capability to handle and select protein complexes that behave problematically in conventional sample preparation, the approach has the potential to expand the scope of both native mass spectrometry and cryo-EM.

Imaging conformations of holo- and apo-transferrin on the single-molecule level by low-energy electron holography.

Conformational changes play a key role in the biological function of many proteins, thereby sustaining a multitude of processes essential to life. Thus, the imaging of the conformational space of proteins exhibiting such conformational changes is of great interest. Low-energy electron holography (LEEH) in combination with native electrospray ion beam deposition (ES-IBD) has recently been demonstrated to be capable of exploring the conformational space of conformationally highly variable proteins on the single-molecule level. While the previously studied conformations were induced by changes in environment, it is of relevance to assess the performance of this imaging method when applied to protein conformations inherently tied to a function-related conformational change. We show that LEEH imaging can distinguish different conformations of transferrin, the major iron transport protein in many organisms, by resolving a nanometer-scale cleft in the structure of the iron-free molecule (apo-transferrin) resulting from the conformational change associated with the iron binding/release process. This, along with a statistical analysis of the data, which evidences a degree of flexibility of the molecules, indicates that LEEH is a viable technique for imaging function-related conformational changes in individual proteins.

Cryo-EM samples of gas-phase purified protein assemblies using native electrospray ion-beam deposition.

An increasing number of studies on biomolecular function indirectly combine mass spectrometry (MS) with imaging techniques such as cryo electron microscopy (cryo-EM). This approach allows information on the homogeneity, stoichiometry, shape, and interactions of native protein complexes to be obtained, complementary to high-resolution protein structures. We have recently demonstrated TEM sample preparation via native electrospray ion-beam deposition (ES-IBD) as a direct link between native MS and cryo-EM. This workflow forms a potential new route to the reliable preparation of homogeneous cryo-EM samples and a better understanding of the relation between native solution-phase and native-like gas-phase structures. However, many aspects of the workflow need to be understood and optimized to obtain performance comparable to that of state-of-the-art cryo-EM. Here, we expand on the previous discussion of key factors by probing the effects of substrate type and deposition energy. We present and discuss micrographs from native ES-IBD samples with amorphous carbon, graphene, and graphene oxide, as well as landing energies in the range between 2 and 150 eV per charge.

A Preparative Mass Spectrometer to Deposit Intact Large Native Protein Complexes.

Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase, where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information on well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific Q Exactive UHMR mass spectrometer). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential.

Mass-selective and ice-free electron cryomicroscopy protein sample preparation via native electrospray ion-beam deposition.

Despite tremendous advances in sample preparation and classification algorithms for electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA), sample heterogeneity remains a major challenge and can prevent access to high-resolution structures. In addition, optimization of preparation conditions for a given sample can be time-consuming. In the current work, it is demonstrated that native electrospray ion-beam deposition (native ES-IBD) is an alternative, reliable approach for the preparation of extremely high-purity samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, separated from other proteins, contaminants, aggregates, and fragments, gently deposited on cryo-EM grids, frozen in liquid nitrogen, and subsequently imaged by cryo-EM. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected protein complexes. SPA reveals that the complexes remain folded and assembled, but variations in secondary and tertiary structures are currently limiting information in 2D classes and 3D EM density maps. We identify and discuss challenges that need to be addressed to obtain a resolution comparable to that of the established cryo-EM workflow. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM for protein structure determination and provide an essential link between gas-phase and solution-phase protein structures.

NaViA: a program for the visual analysis of complex mass spectra.

MOTIVATION: Native mass spectrometry is now a well-established method for the investigation of protein complexes, specifically their subunit stoichiometry and ligand binding properties. Recent advances allowing the analysis of complex mixtures lead to an increasing diversity and complexity in the spectra obtained. These spectra can be time-consuming to tackle through manual assignment and challenging for automated approaches. RESULTS: Native Mass Spectrometry Visual Analyser is a web-based tool to augment the manual process of peak assignment. In addition to matching masses to the stoichiometry of its component subunits, it allows raw data processing, assignment and annotation and permits mass spectra to be shared with their respective interpretation. AVAILABILITY AND IMPLEMENTATION: NaViA is open-source and can be accessed online under https://navia.ms. The source code and documentation can be accessed at https://github.com/d-que/navia, under the BSD 2-Clause licence. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Electronic Action Spectroscopy on Single Nanoparticles in the Gas Phase.

We present electronic excitation spectra of individual nanoparticles (NPs) in the gas phase obtained by messenger-mediated single nanoparticle action spectroscopy at cryogenic temperatures (cryo-SNAS). Single ∼100 nm diameter SiO2 NPs, either colorless or dye-loaded, are trapped and coated with multiple layers of N2 in a temperature-controllable modified quadrupole ion-trap at 100 K. The NP's mass is monitored quasi-continuously and nondestructively by light scattering. Absorption of electromagnetic radiation from a tunable (400-800 nm), quasi-continuous, supercontinuum laser leads to heating of the NP and subsequent evaporation of N2 molecules. The average change in NP mass as a function of the irradiation wavelength then yields the cryo-SNAS spectrum without further correction. The obtained spectra are similar to direct absorption spectra of the corresponding NP suspensions but reveal narrower bands due to the lower NP temperature. These experiments demonstrate that cryo-SNAS allows the determination of photoabsorption spectra of single, free NPs independently of scattering processes.

A cryogenic single nanoparticle action spectrometer.

A nanoparticle (NP) mass spectrometer designed to perform action spectroscopy on single NPs at cryogenic temperatures is described. NPs from an electrospray ion source with masses ranging from 460 to 740 MDa are injected and trapped in a temperature controllable (8-350 K) split-ring electrode ion-trap characterized by improved optical access and trapping potential. After excess NPs are ejected from the trap, the mass-to-charge ratio and subsequently the absolute mass of the trapped NP are determined nondestructively using Fourier transformation and resonant excitation methods. The setup allows us to monitor the mass variation of a single NP as a function of the ion-trap temperature, collision-gas pressure, and irradiation laser power. Ion-trap temperature controlled N2 adsorption at cryogenic temperatures onto a single, ∼90 nm diameter SiO2 NP is demonstrated and characterized. We further show that laser irradiation at 532 nm leads to power-dependent changes in the effective N2 adsorption rate of the particle, which can be monitored and ultimately exploited to measure absorption spectra of a single NP.

Influence of argon and D2 tagging on the hydrogen bond network in Cs+(H2O)3; kinetic trapping below 40 K.

The influence of enthalpic and entropic effects as well as of kinetic trapping processes on the structure of Ar/D2-tagged Cs+(H2O)3 clusters is studied by temperature-dependent infrared photodissociation spectroscopy combined with harmonic vibrational spectra calculations and anharmonic free energy profiles from finite temperature metadynamics molecular dynamics simulations. Each tag favors a different hydrogen bond network of water molecules, with Ar-tagging (vs. D2-tagging) of Cs+(H2O)3 leading to the lower energy conformation. The relative population of these conformers can be tuned over a temperature range of 12 to 21 K. The formation mechanisms of these tagged clusters can be deduced from the free energy profiles. This investigation demonstrates that a variety of factors, both thermodynamic and kinetic, play a role in the structure of flexible molecular species, even at cryogenic temperatures.

Deconstructing Prominent Bands in the Terahertz Spectra of H7O3+ and H9O4+: Intermolecular Modes in Eigen Clusters.

We report experimental vibrational action spectra (210-2200 cm-1) and calculated IR spectra, using recent ab initio potential energy and dipole moment surfaces, of H7O3+ and H9O4+. We focus on prominent far-IR bands, which postharmonic analyses show, arise from two types of intermolecular motions, i.e., hydrogen bond stretching and terminal water wagging modes, that are similar in both clusters. The good agreement between experiment and theory further validates the accuracy of the potential and dipole moment surfaces, which was used in a recent theoretical study that concluded that infrared photodissociation spectra of the cold clusters correspond to the Eigen isomer. The comparison between theory and experiment adds further confirmation of the need of postharmonic analysis for these clusters.

Gas phase vibrational spectroscopy of the protonated water pentamer: the role of isomers and nuclear quantum effects.

We use cryogenic ion trap vibrational spectroscopy to study the structure of the protonated water pentamer, H+(H2O)5, and its fully deuterated isotopologue, D+(D2O)5, over nearly the complete infrared spectral range (220-4000 cm-1) in combination with harmonic and anharmonic electronic structure calculations as well as RRKM modelling. Isomer-selective IR-IR double-resonance measurements on the H+(H2O)5 isotopologue establish that the spectrum is due to a single constitutional isomer, thus discounting the recent analysis of the band pattern in the context of two isomers based on AIMD simulations 〈W. Kulig and N. Agmon, Phys. Chem. Chem. Phys., 2014, 16, 4933-4941〉. The evolution of the persistent bands in the D+(D2O)5 cluster allows the assignment of the fundamentals in the spectra of both isotopologues, and the simpler pattern displayed by the heavier isotopologue is consistent with the calculated spectrum for the branched, Eigen-based structure originally proposed 〈J.-C. Jiang, et al., J. Am. Chem. Soc., 2000, 122, 1398-1410〉. This pattern persists in the vibrational spectra of H+(H2O)5 in the temperature range from 13 K up to 250 K. The present study also underscores the importance of considering nuclear quantum effects in predicting the kinetic stability of these isomers at low temperatures.

Thermodynamics of water dimer dissociation in the primary hydration shell of the iodide ion with temperature-dependent vibrational predissociation spectroscopy.

The strong temperature dependence of the I(-)·(H2O)2 vibrational predissociation spectrum is traced to the intracluster dissociation of the ion-bound water dimer into independent water monomers that remain tethered to the ion. The thermodynamics of this process is determined using van't Hoff analysis of key features that quantify the relative populations of H-bonded and independent water molecules. The dissociation enthalpy of the isolated water dimer is thus observed to be reduced by roughly a factor of three upon attachment to the ion. The cause of this reduction is explored with electronic structure calculations of the potential energy profile for dissociation of the dimer, which suggest that both reduction of the intrinsic binding energy and vibrational zero-point effects act to weaken the intermolecular interaction between the water molecules in the first hydration shell. Additional insights are obtained by analyzing how classical trajectories of the I(-)·(H2O)2 system sample the extended potential energy surface with increasing temperature.

Site-specific vibrational spectral signatures of water molecules in the magic H3O+ (H2O)20 and Cs+ (H2O)20 clusters.

Theoretical models of proton hydration with tens of water molecules indicate that the excess proton is embedded on the surface of clathrate-like cage structures with one or two water molecules in the interior. The evidence for these structures has been indirect, however, because the experimental spectra in the critical H-bonding region of the OH stretching vibrations have been too diffuse to provide band patterns that distinguish between candidate structures predicted theoretically. Here we exploit the slow cooling afforded by cryogenic ion trapping, along with isotopic substitution, to quench water clusters attached to the H3O(+) and Cs(+) ions into structures that yield well-resolved vibrational bands over the entire 215- to 3,800-cm(-1) range. The magic H3O(+)(H2O)20 cluster yields particularly clear spectral signatures that can, with the aid of ab initio predictions, be traced to specific classes of network sites in the predicted pentagonal dodecahedron H-bonded cage with the hydronium ion residing on the surface.

Spectroscopic identification of a bidentate binding motif in the anionic magnesium-CO2 complex ([ClMgCO2 ](-)).

A magnesium complex incorporating a novel metal-CO2 binding motif is spectroscopically identified. Here we show with the help of infrared photodissociation spectroscopy that the complex exists solely in the [ClMg(η(2) -O2 C)](-) form. This bidentate double oxygen metal-CO2 coordination has previously not been observed in neutral nor in charged unimetallic complexes. The antisymmetric CO2 stretching mode in [ClMg(η(2) -O2 C)](-) is found at 1128 cm(-1) , which is considerably redshifted from the corresponding mode in bare CO2 at 2349 cm(-1) , suggesting that the CO2 moiety has a considerable negative charge (∼1.8 e(-) ). We also employed electronic structure calculations and kinetic analysis to support the interpretation of the experimental results.