Low-energy electron holography imaging of conformational variability of single-antibody molecules from electrospray ion beam deposition

Imaging of proteins at the single-molecule level can reveal conformational variability, which is essential for the understanding of biomolecules. To this end, a biologically relevant state of the sample must be retained during both sample preparation and imaging. Native electrospray ionization (ESI) can transfer even the largest protein complexes into the gas phase while preserving their stoichiometry and overall shape. High-resolution imaging of protein structures following native ESI is thus of fundamental interest for establishing the relation between gas phase and solution structure. Taking advantage of low-energy electron holography’s (LEEH) unique capability of imaging individual proteins with subnanometer resolution, we investigate the conformational flexibility of Herceptin, a monoclonal IgG antibody, deposited by native electrospray mass-selected ion beam deposition (ES-IBD) on graphene. Images reconstructed from holograms reveal a large variety of conformers. Some of these conformations can be mapped to the crystallographic structure of IgG, while others suggest that a compact, gas-phase–related conformation, adopted by the molecules during ES-IBD, is retained. We can steer the ratio of those two types of conformations by changing the landing energy of the protein on the single-layer graphene surface. Overall, we show that LEEH can elucidate the conformational heterogeneity of inherently flexible proteins, exemplified here by IgG antibodies, and thereby distinguish gas-phase collapse from rearrangement on surfaces.

Mass-selective and ice-free cryo-EM protein sample preparation via native electrospray ion-beam deposition

Electron cryomicroscopy (cryo-EM) and single-particle analysis (SPA) have revolutionized structure determination of homogeneous proteins. However, obtaining high-resolution structures from heterogeneous samples remains a major challenge, as the various protein states embedded in thin films of vitreous ice may be classified incorrectly, resulting in detrimental averaging of features. Here we present native electrospray ion-beam deposition (native ES-IBD) for the preparation of extremely high-purity cryo-EM samples, based on mass selection in vacuum. Folded protein ions are generated by native electrospray ionization, mass-filtered, and gently deposited on cryo-EM grids, and subsequently frozen in liquid nitrogen. We demonstrate homogeneous coverage of ice-free cryo-EM grids with mass-selected proteins and protein assemblies. SPA reveals that they remain structurally intact, but variations in secondary and tertiary structure are currently limiting information in 2D classes and 3D EM density maps. Our results show the potential of native ES-IBD to increase the scope and throughput of cryo-EM structure determination.

Navigate flying molecular elephants safely to the ground: mass-selective soft landing up to the Mega-Dalton range by Electrospray Controlled Ion-Beam Deposition

The prototype of a highly versatile and efficient preparative mass spectrometry system used for the deposition of molecules in ultra-high vacuum (UHV) is presented, along with encouraging performance data obtained on four model species which are thermolabile or not sublimable. The test panel comprises two small organic compounds, a protein, and a large DNA species covering a 4-log mass range up to 1.7 MDa as part of a broad spectrum of analyte species evaluated to date. Three designs of innovative ion guides, a novel digital mass-selective quadrupole (dQMS) and a standard electrospray ionization (ESI) source are combined to an integrated device, abbreviated Electrospray Controlled Ion Beam Deposition (ES-CIBD). Full control is achieved by i) the square-wave-driven radiofrequency (RF) ion guides with steadily tunable frequencies, including a dQMS allowing for investigation, purification and deposition of a virtually unlimited m/z range, ii) the adjustable landing energy of ions down to ~2 eV/z enabling integrity-preserving soft-landing, iii) the deposition in UHV with high ion beam intensity (up to 3 nA) limiting contaminations and deposition time, and iv) direct coverage control via the deposited charge. The maximum resolution of R=650 and overall efficiency up to T_total=4.4% calculated from solution to UHV deposition are remarkable as well, while the latter is mainly limited by the not yet optimized ionization performance. In the setup presented, a scanning tunneling microscope (STM) is attached for in situ UHV investigation of the deponents demonstrating a selective, structure-preserving process and atomically clean layers.

Navigate flying molecular elephants safely to the ground: mass-selective soft landing up to the Mega-Dalton range by Electrospray Controlled Ion-Beam Deposition

The prototype of a highly versatile and efficient preparative mass spectrometry system used for the deposition of molecules in ultra-high vacuum (UHV) is presented along with encouraging performance data obtained on model species which are thermolabile or not sublimable. The test panel comprises two organic compounds, a protein, and DNA covering a 4-log mass range up to 1.7 MDa as part of a broad spectrum of analyte species. Three designs of innovative ion guides, a novel digital mass-selective quadrupole (dQMS) and a standard electrospray ionization (ESI) source are combined to an integrated device, abbreviated Electrospray Controlled Ion Beam Deposition (ES-CIBD). Full control is achieved by i) the square-wave-driven radiofrequency (RF) ion guides with steadily tunable frequencies, including a dQMS allowing for investigation, purification and deposition of a virtually unlimited m/z range, ii) the adjustable landing energy of ions down to ~2 eV/z enabling integrity-preserving soft-landing, iii) the deposition in UHV with high ion beam intensity (up to 3 nA) limiting contaminations and deposition time, and iv) direct coverage control via the deposited charge. The maximum resolution of R=650 and overall efficiency up to T-total=4.4% calculated from solution to UHV deposition are remarkable as well, while the latter is mainly limited by the not yet optimized ionization performance. In the setup presented, a scanning tunneling microscope (STM) is attached for in situ UHV investigation of the deponents demonstrating a selective, structure-preserving process and atomically clean layers.

Influence of the autoirradiation on nanosized gold layers formation by the ion-beam deposition

2–13 nm gold films were obtained by the method of ion-beam sputtering on silicon and quartz substrates. It is shown that the use of an additional operation of deposition followed by the sputtering of a gold layer of 2–3 nm thickness makes it possible to reduce the electrical resistance and surface roughness of the metal films, in comparison with similar films obtained without its use. The results of measuring the temperature coefficient of resistance of nanosized gold films on silicon substrates allowed us to conclude that the films deposited become continuous at a thickness of 6-8 nm. The results of optical measurements of 10 nm gold films, obtained on quartz substrates, showed that the reflection coefficient of electromagnetic radiation at a wavelength of 850 nm is 2.8 % higher than the corresponding coefficient for the same films obtained without using this operation, and is 83 %. An important role in the formation of nanoscale gold layers is played by the processes of self-irradiation of the growing layer of the high-energy component of the gold atoms flux. When using an additional operation of deposition/sputtering, high-energy gold atoms are implanted into the substrate to a depth of about 2 nm. On the one hand, these atoms are point defects in the surface damaged layer of the substrate; on the other hand, they serve as additional centers of cluster formation. This ensures strong adhesion of the metal layer to the substrate and, therefore, the gold films become continuous and more homogeneous in microstructure. The method of ion-beam deposition can be successfully applied to obtain high-quality conductive optically transparent nanosized gold films.

Monitoring Carbon in Electron and Ion Beam Deposition within FIB-SEM

It is well known that carbon present in scanning electron microscopes (SEM), Focused ion beam (FIB) systems and FIB-SEMs, causes imaging artefacts and influences the quality of TEM lamellae or structures fabricated in FIB-SEMs. The severity of such effects depends not only on the quantity of carbon present but also on its bonding state. Despite this, the presence of carbon and its bonding state is not regularly monitored in FIB-SEMs. Here we demonstrated that Secondary Electron Hyperspectral Imaging (SEHI) can be implemented in different FIB-SEMs (ThermoFisher Helios G4-CXe PFIB and Helios Nanolab G3 UC) and used to observe carbon built up/removal and bonding changes resulting from electron/ion beam exposure. As well as the ability to monitor, this study also showed the capability of Plasma FIB Xe exposure to remove carbon contamination from the surface of a Ti6246 alloy without the requirement of chemical surface treatments.