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Significance Molecular imaging at the single-molecule level of large and flexible proteins such as monoclonal IgG antibodies is possible by low-energy electron holography after chemically selective sample preparation by native electrospray ion beam deposition (ES-IBD) from native solution conditions. The single-molecule nature of the measurement allows the mapping of the structural variability of the molecules that originates from their intrinsic flexibility and from different adsorption geometries. Additionally, we can distinguish gas-phase–related conformations and conformations induced by the landing of the molecules on the surface. Our results underpin the relation between the gas-phase structure of protein ions created by native electrospray ionization (ESI) and the native protein structure and are of relevance for structural biology applications in the gas phase., 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.

Thin inorganic films (i.e., metal oxides) are often used as decoupling layers to optimize the interactions between the magnetic layers of molecules and metallic surfaces. For deposits of single-molecule magnets (SMMs), a decoupling layer can minimise the hybridization of the metallic substrate that is responsible for the quenching of their typical magnetic bistability. Here, we explored the potential of a single layer of titania to be used as a decoupling layer, which could represent an interesting playground for widespread use in many technological applications. We used a TiO2 monolayer with a lepidocrocite-like structure grown on a Ag(100) substrate for the deposition of the terbium(iii) bis-phthalocyaninato (TbPc2) complex. A multi-technique approach employing X-ray photoelectron spectroscopy and scanning tunnelling microscopy was used to examine the integrity of a TbPc2 sub-monolayer deposit and to study the molecular adsorption configuration on the TiO2 film. Furthermore, X-ray magnetic circular dichroism was used to investigate the magnetic properties of the TbPc2 sub-monolayer, revealing that the TiO2 film successfully preserves the molecular spin character. X-ray-based magnetic measurements showed that the quantum tunnelling of the magnetization characterizing a bulk of molecules is still present and that the present titania film displays a decoupling effect of comparable efficiency to that of a graphene layer.