Charting the molecular landscape of the cell.

Biological function of macromolecules is closely tied to their cellular location, as well as to interactions with other molecules within the native environment of the cell. Therefore, to obtain detailed mechanistic insights into macromolecular functionality, one of the outstanding targets for structural biology is to produce an atomic-level understanding of the cell. One structural biology technique that has already been used to directly derive atomic models of macromolecules from cells, without any additional external information, is electron cryotomography (cryoET). In this perspective article, we discuss possible routes to chart the molecular landscape of the cell by advancing cryoET imaging as well as by embedding cryoET into correlative imaging workflows.

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.

Electrospray ion beam deposition plus low-energy electron holography as a tool for imaging individual biomolecules.

Inline low-energy electron holography (LEEH) in conjunction with sample preparation by electrospray ion beam deposition (ES-IBD) has recently emerged as a promising method for the sub-nanometre-scale single-molecule imaging of biomolecules. The single-molecule nature of the LEEH measurement allows for the mapping of the molecules' conformational space and thus for the imaging of structurally variable biomolecules, thereby providing valuable complementary information to well-established biomolecular structure determination methods. Here, after briefly tracing the development of inline LEEH in bioimaging, we present the state-of-the-art of native ES-IBD + LEEH as a method of single-protein imaging, discuss its applications, specifically regarding the imaging of structurally flexible protein systems and the amplitude and phase information encoded in a low-energy electron hologram, and provide an outlook regarding the considerable possibilities for the future advancement of the approach.

Landing Proteins on Graphene Trampoline Preserves Their Gas-Phase Folding on the Surface.

Molecule-surface collisions are known to initiate dynamics that lead to products inaccessible by thermal chemistry. These collision dynamics, however, have mostly been examined on bulk surfaces, leaving vast opportunities unexplored for molecular collisions on nanostructures, especially on those that exhibit mechanical properties radically different from those of their bulk counterparts. Probing energy-dependent dynamics on nanostructures, particularly for large molecules, has been challenging due to their fast time scales and high structural complexity. Here, by examining the dynamics of a protein impinging on a freestanding, single-atom-thick membrane, we discover molecule-on-trampoline dynamics that disperse the collision impact away from the incident protein within a few picoseconds. As a result, our experiments and ab initio calculations show that cytochrome c retains its gas-phase folded structure when it collides onto freestanding single-layer graphene at low energies (∼20 meV/atom). The molecule-on-trampoline dynamics, expected to be operative on many freestanding atomic membranes, enable reliable means to transfer gas-phase macromolecular structures onto freestanding surfaces for their single-molecule imaging, complementing many bioanalytical techniques.

Phase Reconstruction of Low-Energy Electron Holograms of Individual Proteins.

Low-energy electron holography (LEEH) is one of the few techniques capable of imaging large and complex three-dimensional molecules, such as proteins, on the single-molecule level at subnanometer resolution. During the imaging process, the structural information about the object is recorded both in the amplitude and in the phase of the hologram. In low-energy electron holography imaging of proteins, the object's amplitude distribution, which directly reveals molecular size and shape on the single-molecule level, can be retrieved via a one-step reconstruction process. However, such a one-step reconstruction routine cannot directly recover the phase information encoded in the hologram. In order to extract the full information about the imaged molecules, we thus implemented an iterative phase retrieval algorithm and applied it to experimentally acquired low-energy electron holograms, reconstructing the phase shift induced by the protein along with the amplitude data. We show that phase imaging can map the projected atomic density of the molecule given by the number of atoms in the electron path. This directly implies a correlation between reconstructed phase shift and projected mean inner potential of the molecule, and thus a sensitivity to local changes in potential, an interpretation that is further substantiated by the strong phase signatures induced by localized charges.

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.

Catalyzing Bond-Dissociation in Graphene via Alkali-Iodide Molecules.

Atomic design of a 2D-material such as graphene can be substantially influenced by etching, deliberately induced in a transmission electron microscope. It is achieved primarily by overcoming the threshold energy for defect formation by controlling the kinetic energy and current density of the fast electrons. Recent studies have demonstrated that the presence of certain species of atoms can catalyze atomic bond dissociation processes under the electron beam by reducing their threshold energy. Most of the reported catalytic atom species are single atoms, which have strong interaction with single-layer graphene (SLG). Yet, no such behavior has been reported for molecular species. This work shows by experimentally comparing the interaction of alkali and halide species separately and conjointly with SLG, that in the presence of electron irradiation, etching of SLG is drastically enhanced by the simultaneous presence of alkali and iodine atoms. Density functional theory and first principles molecular dynamics calculations reveal that due to charge-transfer phenomena the CC bonds weaken close to the alkali-iodide species, which increases the carbon displacement cross-section. This study ascribes pronounced etching activity observed in SLG to the catalytic behavior of the alkali-iodide species in the presence of electron irradiation.

Fast Molecular Compression by a Hyperthermal Collision Gives Bond-Selective Mechanochemistry.

Using electrospray ion beam deposition, we collide the 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 (subpicosecond) is much shorter than the thermalization timescale (nanosecond), thereby yielding reaction products that are unobtainable thermally.

Substrate-Selective Morphology of Cesium Iodide Clusters on Graphene.

Formation and characterization of low-dimensional nanostructures is crucial for controlling the properties of two-dimensional (2D) materials such as graphene. Here, we study the structure of low-dimensional adsorbates of cesium iodide (CsI) on free-standing graphene using aberration-corrected transmission electron microscopy at atomic resolution. CsI is deposited onto graphene as charged clusters by electrospray ion-beam deposition. The interaction with the electron beam forms two-dimensional CsI crystals only on bilayer graphene, while CsI clusters consisting of 4, 6, 7, and 8 ions are exclusively observed on single-layer graphene. Chemical characterization by electron energy-loss spectroscopy imaging and precise structural measurements evidence the possible influence of charge transfer on the structure formation of the CsI clusters and layers, leading to different distances of the Cs and I to the graphene.