Cryo-EM of soft-landed β-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 β-galactosidase using single-particle cryo-electron microscopy (cryo-EM) down to 2.

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.

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 native electrospray ion-beam deposition (ES-IBD) as a direct link between native MS and cryo-EM.

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.

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.

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 SiO NPs, either colorless or dye-loaded, are trapped and coated with multiple layers of N in a temperature-controllable modified quadrupole ion-trap at 100 K. The NP's mass is monitored quasi-continuously and nondestructively by light scattering.

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.

Influence of argon and D tagging on the hydrogen bond network in Cs(HO); 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.

Deconstructing Prominent Bands in the Terahertz Spectra of HO and HO: Intermolecular Modes in Eigen Clusters.

We report experimental vibrational action spectra (210-2200 cm) and calculated IR spectra, using recent ab initio potential energy and dipole moment surfaces, of HO and HO. We focus on prominent far-IR bands, which postharmonic analyses show, arise from two types of intermolecular motions, i.e.

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(HO), and its fully deuterated isotopologue, D(DO), over nearly the complete infrared spectral range (220-4000 cm) in combination with harmonic and anharmonic electronic structure calculations as well as RRKM modelling. Isomer-selective IR-IR double-resonance measurements on the H(HO) 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.

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.

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.

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.