Imaging proteins at the single-molecule level

Abstract

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 ...

Von isolierten Ionen zu mehrschichtigen funktionellen Materialien durch sanfte Landung von Ionen

“…[85] Bei diesem Ansatz wurden Proteine auf Graphen abgelagert und mit Niedrigenergie-Elektronenholographie untersucht. Kürzlich wurde eine neue Te chnik zum Abbilden von sanft gelandeten Proteinen in ihrer natürlichen Konformation vorgestellt.…”

Nanofocused Plasmon-Driven Sub-10 fs Electron Point Source

“…By reducing the tip-sample distance to the sub-µm range, the purely geometric projection transforms into a hologram and fsPPM merges into femtosecond low-energy electron in-line holography. In-line holographic imaging of individual biological specimen with 1 nm spatial resolution at the anode has been realized recently [20,52] by using graphene [19] as sample support, thus reducing the biprism effect which is detrimental if high spatial resolution is desired [53]. Beyond such sample restrictions, the spatial resolution of femtosecond in-line holography will ultimately be determined by the spatial coherence of the electron source, which is given by the effective source size r eff and the electron energy spread [54].…”

Expanding the structural biology toolbox with single-molecule holography

“…Thanks to mechanical robustness combined with excellent transparency to electron beams, graphene supports have already been proven excellent for bioimaging and are also being considered for advanced cryo-EM (16-18). Longchamp et al (13) developed an efficient method to generate ultra-clean graphene supports, which is now fully implemented in the experimental pipeline (19). To achieve contamination-free transfer of graphene grids from the electrospray device to the holography microscope, the authors used custom-designed suitcases that allow portable 10 −10 vacuum levels.…”

“…Unambiguous real-space reconstruction of visualized macromolecules can be performed immediately after recording using mathematical reconstructions (23)(24)(25). Strikingly, Longchamp et al (13) report that neutralization of protein ions spontaneously occurs when deposited on graphene. This important point must be thoroughly investigated because charges on the molecule surface may act as lenses that effectively deviate the low-energy electron beams, interfering with real-space reconstruction of the object under observation.…”

“…In addition, Longchamp et al (13) report that data collections at different times do not show alterations, except for motions of the sample on the graphene surface, indicating a complete absence of radiation damage.…”