Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical-and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process. low-energy electron holography | single protein imaging | preparative mass spectrometry | microscopy | structural biology M ost of the currently available information on structures of macromolecules and proteins has been obtained from either X-ray crystallography experiments or cryo-electron microscopy investigations by means of averaging over many molecules assembled into a crystal or over a large ensemble selected from low signal-tonoise ratio electron micrographs, respectively (1). Despite the impressive coverage of the proteome by the available data, a strong desire for acquiring structural information from just one individual molecule is emerging. The biological relevance of a protein lies in its structural dynamics, which are accompanied by distinct conformations. For a protein to fulfill its vital functions in a living organism, it cannot exist in just one single and fixed structure, but needs to be able to assume different conformations to carry out specific functions. Conceptually, at least two different conformations, just like in a simple switch, are needed. In view of oxygen transport to cells for example, binding oxygen in one specific conformation and releasing it again in a different conformation are needed. To address the "physics of proteins" as described by Hans Frauenfelder in his pioneering review (2), one needs to realize that proteins are complex systems assuming different conformations and exhibiting a rich free-energy landscape. The associated structural details, however, remain undiscovered when averaging is involved. Moreover, a large subset of the entirety of proteins, in particular from the important category of membrane proteins, is extremely difficult, if not impossible, to obtain in a crystalline form. If just one individual protein or protein complex can be analyzed in sufficient detail, those objects will finally become accessible.For a meaningful contribution to structural biology, a tool for single-molecule imaging must allow for observing an individual protein long enough to acquire a sufficient amount of data to reveal its structure without altering it. The strong inelastic scattering cross-section of high-energy ...

Ionic bonding in supramolecular surface networks is a promising strategy to self-assemble nanostructures from organic building blocks with atomic precision. However, sufficient thermal stability of such systems has not been achieved at metal surfaces, likely due to partial screening of the ionic interactions. We demonstrate excellent stability of a self-assembled ionic network on a metal surface at elevated temperatures. The structure is characterized directly by atomic resolution scanning tunneling microscopy (STM) experiments conducted at 165 °C showing intact domains. This robust nanometer-scale structure is achieved by the on-surface reaction of a simple and inexpensive compound, sodium chloride, with a model system for carboxylate interactions, terephthalic acid (TPA). Rather than distinct layers of TPA and NaCl, angle resolved X-ray photoelectron spectroscopy experiments indicate a replacement reaction on the Cu(100) surface to form Na-carboxylate ionic bonds. Chemical shifts in core level electron states confirm a direct interaction and a +1 charge state of the Na. High-temperature STM imaging shows virtually no fluctuation of Na-TPA island boundaries, revealing a level of thermal stability that has not been previously achieved in noncovalent organic-based nanostructures at surfaces. Comparable strength of intermolecular ionic bonds and intramolecular covalent bonds has been achieved in this surface system. The formation of these highly ordered structures and their excellent thermal stability is dependent on the interplay of adsorbate-substrate and ionic interactions and opens new possibilities for ionic self-assemblies at surfaces with specific chemical function. Robust ionic surface structures have potential uses in technologies requiring high thermal stability and precise ordering through self-assembly.

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The sequence of a peptide programs its self-assembly and hence the expression of specific properties through non-covalent interactions. A large variety of peptide nanostructures has been designed employing different aspects of these non-covalent interactions, such as dispersive interactions, hydrogen bonding or ionic interactions. Here we demonstrate the sequence-controlled fabrication of molecular nanostructures using peptides as bio-organic building blocks for two-dimensional (2D) self-assembly. Scanning tunnelling microscopy reveals changes from compact or linear assemblies (angiotensin I) to long-range ordered, chiral honeycomb networks (angiotensin II) as a result of removal of steric hindrance by sequence modification. Guided by our observations, molecular dynamic simulations yield atomistic models for the elucidation of interpeptide-binding motifs. This new approach to 2D self-assembly on surfaces grants insight at the atomic level that will enable the use of oligo- and polypeptides as large, multi-functional bio-organic building blocks, and opens a new route towards rationally designed, bio-inspired surfaces.

We report our investigation on the nanorods of two newly synthesized substituted pentacenes, d 4 -substituted (2,3-X 2 -9,10-Y 2 ) pentacene with X = Y = methoxy group (MOP) and X = F, Y = methoxy (MOPF), by using X-ray photoemission spectroscopy (XPS), near edge X-ray absorption fine structure (NEXAFS), and atomic force microscopy (AFM). The nanorods were deposited on Au(111) single crystals. Energy dependent photoemission spectra show complex features, including a rich satellite structure that we have analyzed in detail by using a bestfit procedure applying constraints based on stoichiometry, electronegativity, and bond strength. This analysis reveals the presence of surface core level shifts due to the high electronegativity of the fluorine atoms. The distinctive features of growth and morphology of the nanorods are subjected to a template effect by the substrate lattice geometry, leading to morphological well-organized assemblies. Fluorine atoms play an important role not only in the electronic structure but also in the morphology of the nanorod assemblies.

Biomolecules function by adopting multiple conformations. Such dynamics are governed by the conformation landscape whose study requires characterization of the ground and excited conformation states. Here, the conformational landscape of a molecule is sampled by exciting an initial gas-phase molecular conformer into diverse conformation states, using soft molecule–surface collision (0.5–5.0 eV). The resulting ground and excited molecular conformations, adsorbed on the surface, are imaged at the single-molecule level. This technique permits the exploration of oligosaccharide conformations, until now, limited by the high flexibility of oligosaccharides and ensemble-averaged analytical methods. As a model for cellulose, cellohexaose chains are observed in two conformational extremes, the typical “extended” chain and the atypical “coiled” chain—the latter identified as the gas-phase conformer preserved on the surface. Observing conformations between these two extremes reveals the physical properties of cellohexaose, behaving as a rigid ribbon that becomes flexible when twisted. The conformation space of any molecule that can be electrosprayed can now be explored.

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.

Using electrospray ion beam deposition, we collide a complex molecule Reichardt's Dye (C 41 H 30 NO + ) at low, hyperthermal translational energy (2 -50 eV) with a Cu(100) surface and image the outcome at single-molecule level by Scanning Tunneling Microscopy. We observe bond-selective reaction induced by the translational kinetic energy. The collision impulse compresses the molecule and bends specific bonds, prompting them to react selectively. This dynamics drives the system to seek thermally inaccessible reactive pathways, since the compression timescale (sub-ps) is much shorter than the thermalization timescale (ns), thereby yielding reaction products that are unobtainable thermally