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.

Keywords: low-energy electron holography; microscopy; preparative mass spectrometry; single protein imaging; structural biology.

Fig. 1.

Schematic workflow for imaging a single protein. Left to Right: An ultraclean graphene sample is characterized in a low-energy electron holographic microscope. Shown is deposition of proteins onto freestanding graphene in an m/z filtered ES-IBD system. Imaging of the proteins within the previously characterized region by means of low-energy electron holography. An electron point source (EPS) emits a divergent beam of highly coherent low-energy electrons and holograms of the deposited proteins are recorded on the detector. Throughout the experimental workflow, the sample is kept under strict UHV conditions with the help of an UHV suitcase for the transfer between the two experimental chambers (UHV Transfer for more details).

Fig. S1.

Illustration of the working principle of the home-built instrument for soft-landing electrospray ion beam deposition of proteins. The proteins are directly deposited from the liquid phase onto a substrate kept at UHV conditions to avoid contaminations.

Fig. S2.

Mass spectra of CytC (Top), BSA (Middle), and hemoglobin (Bottom). (Left) The m/z spectra before mass filtering are displayed. (Right) The corresponding mass-filtered spectra.

Fig. 2.

Low-energy electron holograms of CytC and their reconstructions. (A–C) Three holograms of CytC. (D–F) Numerical reconstructions revealing the shapes of the objects. The diffuse rings around the object are due to the presence of the out-of-focus twin image inherent to in-line holography. (Scale bars, 2 nm.)

Fig. S3.

Low-energy electron holography scheme.

Fig. 3.

Complete dataset for the imaging of CytC. (A) Low-energy electron image of ultraclean graphene covering a 500 × 500-nm² aperture before protein deposition. (B) Mass spectrum of the mass-selected CytC beam. (C) A survey image of the very same freestanding graphene region after deposition of CytC. (D–J) Low-energy electron micrographs with suggestions for possible protein orientations based on the averaged protein structure derived from X-ray crystallography data and documented in the PDB (PDB ID: 1HRC). (Scale bars, 2 nm.)

Fig. S4.

UHV vacuum suitcase and its performance. (A) Three-dimensional rendering of the transport suitcase enabling UHV transfer of ultraclean freestanding graphene between the low-energy electron holographic microscope and the ES-IBD chamber. (B and C) Low-energy electron projection images before (B) and after (C) a complete transfer and travel cycle without protein deposition. In the course of the transfer procedure between the two vacuum chambers no relevant contamination of the graphene has built up. (Scale bar, 100 nm.)

Fig. 4.

Low-energy electron micrographs of BSA in different orientations on graphene. (Top) Low-energy electron micrographs of BSA. (Bottom) The atomic model of BSA (PDB ID: 3V03) in the corresponding orientations. (Scale bars, 5 nm.)

Fig. 5.

Comparison of low-energy electron micrographs of BSA with simulated electron density maps. (Top) Low-energy electron micrographs of BSA. (Scale bars, 5 nm.) (Middle) Electron density maps simulated at a resolution of 8 Å and rotated to match the orientation of the proteins presented in Top. (Bottom) Side view of the density map along the directions of observation indicated by the arrows shown in Middle.

Fig. 6.

Low-energy electron micrographs of two individual HG molecules and the atomic model in the corresponding orientations. (Top) Two micrographs of HG soft landed onto freestanding graphene. (Bottom) Suggestions for possible orientations based on the averaged protein structure derived from X-ray crystallography data and documented in the PDB (PDB ID: 2QSS). (Scale bars, 5 nm.)

Fig. S5.

Time evolution of the orientation of CytC complexes. The time lapse between subsequent observations amounts to 30 s. From these images it is evident that at least some of the deposited proteins are mobile on freestanding graphene. Thus, low-energy electron holography appears to be a method for also studying diffusion of proteins on surfaces. This observation suggests that a low-energy electron holographic microscope operating at cryogenic temperatures might be needed to fix the protein in space and attain atomic resolution. (Scale bars, 5 nm.)