Jean-Nicolas Longchamp a,1, Stephan Rauschenbach b, Sabine Abb b, Conrad Escher a, Tatiana Latychevskaia a, Klaus Kern b,c,1, and Hans-Werner Fink a,1
a Physics Department of the University of Zurich, CH-8057 Zurich, Switzerland;
b Max Planck Institute for Solid State Research, DE-70569 Stuttgart, Germany;
c Institut de Physique, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
Edited by R. Graham Cooks, Purdue University, West Lafayette, IN, and approved December 13, 2016 (received for review September 2, 2016)
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.)
We report a method to image and reveal structural details of proteins on a truly single-molecule level. Low-energy electron holography is used to image individual proteins electrospray deposited on freestanding graphene. In contrast to the current state of the art in structural biology, we do away with the need for averaging over many molecules. This is crucial because proteins are flexible objects that can assume distinct conformations often associated with different functions. Proteins are also the targets of almost all the currently known and available drugs. The design of new and more effective drugs relies on the knowledge of the targeted proteins structure in all its biologically significant conformations at the best possible resolution.
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
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Author contributions: J.-N.L. had the original idea to combine ES-IBD and low-energy electron holography and further elaborated the concept with K.K. and H.-W.F. J.-N.L. prepared the ultraclean freestanding graphene supports and recorded the holograms. J.-N.L. and S.R. planned the deposition experiments and along with S.A. performed the electrospray deposition of the proteins onto graphene. T.L. performed the hologram reconstructions with her self-developed software package. J.-N.L. and S.R. interpreted the data. H.-W.F. invented the technology of lens-less holography with low-energy electrons based on atomic sized coherent electron point sources. J.-N.L., C.E., T.L., and H.-W.F. further developed the low-energy electron holographic microscope used in this study. S.R. and K.K. developed the ES-IBD technique. J.-N.L, C.E., and H.-W.F. wrote the manuscript main text and with S.R. the supplementary information, in discussions with all remaining authors.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
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