Design of a molecular support for cryo-EM structure determination Thomas G. Martina, Tanmay A. M. Bharata,b, Andreas C. Joergera,c, Xiao-chen Baia, Florian Praetoriusd, Alan R. Fershta, Hendrik Dietzd,1, and Sjors H. W. Scheresa,1 aMedical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom; bSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom; cGerman Cancer Consortium (DKTK), Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, 60438 Frankfurt am Main, Germany; and dPhysik Department, Walter Schottky Institute, Technische Universität München, 85748 Garching near Munich, Germany Edited by Fred J. Sigworth, Yale University, New Haven, CT, and approved October 13, 2016 (received for review August 2, 2016) Despite the recent rapid progress in cryo-electron microscopy angles are, therefore, determined a posteriori by image-pro- (cryo-EM), there still exist ample opportunities for improvement cessing algorithms that match the experimental projection of in sample preparation. Macromolecular complexes may disassociate every individual particle with projections of a 3D model (7). or adopt nonrandom orientations against the extended air–water However, the projection-matching procedure is ultimately ham- interface that exists for a short time before the sample is frozen. We pered by radiation damage. Because the electrons that are used designed a hollow support structure using 3D DNA origami to pro- for imaging destroy the very structures of interest (see ref. 8 for a tect complexes from the detrimental effects of cryo-EM sample recent review), one needs to limit carefully the number of elec- preparation. For a first proof-of-principle, we concentrated on the trons used for imaging. This procedure results in high levels of transcription factor p53, which binds to specific DNA sequences on experimental noise, which in turn lead to errors in the a poste- double-stranded DNA. The support structures spontaneously form monolayers of preoriented particles in a thin film of water, and offer riori determination of the viewing angles. These errors impose advantages in particle picking and sorting. By controlling the posi- severe limitations on the 3D reconstruction, in particular for tion of the binding sequence on a single helix that spans the hollow smaller complexes, because the signal-to-noise ratio in the im- support structure, we also sought to control the orientation of ages decreases with the size of the particles. If one could ex- individual p53 complexes. Although the latter did not yet yield perimentally control the orientations of each particle in the ice the desired results, the support structures did provide partial in- layer, then, in principle, one could determine structures to higher formation about the relative orientations of individual p53 com- resolution and of smaller complexes. This situation is illustrated plexes. We used this information to calculate a tomographic 3D by samples where the relative orientations of many molecules is reconstruction, and refined this structure to a final resolution of set—for example, in 2D crystals or helical assemblies of protein ∼15 Å. This structure settles an ongoing debate about the symme- molecules. In such cases, near-atomic-resolution reconstructions try of the p53 tetramer bound to DNA. were already achieved decades ago, and from much smaller molecules than currently possible with single-particle analysis (9– cryo-EM | DNA-origami | single particle analysis | structural biology | p53 12). Both developments that triggered the recent revolution in attainable resolution of cryo-EM single-particle analysis directly ryo-electron microscopy (cryo-EM) structure determination addressed this same hurdle. Better detectors led to lower levels Cof biological macromolecules is undergoing rapid progress. With the advent of efficient direct electron detectors and the Significance development of powerful algorithms for image processing, nu- merous structures to near-atomic resolution have been reported As the scope of macromolecular structure determination by in the past few years (1, 2). In cryo-EM single-particle analysis, cryo-electron microscopy (cryo-EM) is expanding rapidly, it is solutions of purified protein and/or nucleic acid complexes are becoming increasingly clear that many biological complexes typically applied to a thin, amorphous carbon film with micro- are too fragile to withstand the harsh conditions involved in meter-sized holes in it that is held in place by a metal grid. Excess making cryo-EM samples. We describe an original approach to liquid is then blotted away with filter paper, and the sample is protect proteins from harmful forces during cryo-EM sample rapidly plunged in liquid ethane (3, 4). This procedure ideally preparation by enclosing them inside a three-dimensional results in the formation of a film of vitreous ice that is only support structure that we designed using DNA origami tech- slightly thicker than the macromolecular complex of interest. niques. By binding the transcription cofactor p53 to a specific Keeping the frozen sample at liquid nitrogen temperatures allows DNA sequence, and by modifying the position of this sequence its insertion into the high vacuum of a transmission electron mi- in our support structure, we also sought to control the relative croscope and limits the effects of radiation damage by the elec- orientation of individual p53:DNA complexes. trons that are used for imaging (5). Images taken through the holes of the carbon film ideally contain 2D projections of many, Author contributions: T.G.M., A.R.F., H.D., and S.H.W.S. designed research; T.G.M., T.A.M.B., assumedly identical copies of the macromolecular complex of and X.-c.B. performed research; A.C.J. and F.P. contributed new reagents/analytic tools; T.G.M., T.A.M.B., X.-c.B., and S.H.W.S. analyzed data; and T.G.M., A.C.J., A.R.F., H.D., and interest, which are often called particles. Projections from dif- S.H.W.S. wrote the paper. ferent viewing directions can then be combined in a 3D re- The authors declare no conflict of interest. construction of the scattering potential of the molecule (6). If the This article is a PNAS Direct Submission. resulting map approaches a resolution of 3 Å, it allows building Freely available online through the PNAS open access option. an atomic model of the molecules, from which useful in- Data deposition: The p53 reconstruction reported in this paper has been deposited in the formation about their function may be derived. Electron Microscopy Data Bank, https://www.ebi.ac.uk/pdbe/emdb (accession no. 3453). A major hurdle in single-particle analysis is the need to re- 1To whom correspondence may be addressed. Email: [email protected] or cover the relative viewing angles of the individual particles. This [email protected]. information is lost in the experiment because every particle This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. adopts an uncontrolled orientation in the ice layer. The viewing 1073/pnas.1612720113/-/DCSupplemental. E7456–E7463 | PNAS | Published online November 7, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1612720113 Downloaded by guest on October 2, 2021 of experimental noise, and better image-processing algorithms Results PNAS PLUS led to more accurate viewing angles. Support Structure Design. Fig. 1 illustrates the design of our pro- Another complication of cryo-EM structure determination lies posed DNA-origami support structure. We designed a hollow in the way the sample is prepared (see also refs. 13 and 14). The pillar with a honey-combed motif of 82 parallel double-stranded exact physics of cryo-EM sample preparation is poorly described, DNA (dsDNA) helices with a height of 26 nm. Two parallel but several factors may negatively affect its results. First, the arrays of DNA helices create a central cavity of 13.2 × 13.6 nm blotting process itself may involve strong forces in the sample and outer dimensions of 26.4 × 32.7 nm. A dsDNA helix with a that destroy fragile protein complexes. Second, during the short central p53-specific binding sequence spans the center of the time between blotting and vitrification, the macromolecules are hollow space. Ten parallel helices at the periphery, which we will in a thin liquid film that extends for millimeters to the side, but is call the flag (Fig. 1 A and B, top left), make the structure asym- only a few hundred angstroms thick. Brownian motion will cause metric so that top and bottom views can be readily distinguished. the macromolecules to collide with the air–water interface Different aspects of this design aim to address four main ob- >1,000 times per second (15). Biological macromolecules may jectives of our support structure. unfold when they hit the air–water interface (16), or they may First, we aimed to keep the target protein in the middle of a adsorb to this interface in a nonrandom manner—for example, sufficiently thin ice layer and away from the air–water interface. by presenting their most hydrophobic patch to it. This interaction p53 binds preferentially to the sequence GGACATGTCCG- leads to an uneven distribution of viewing angles in the cryo-EM GACATGTCC. By including this sequence in the central dsDNA images, and the corresponding lack of different views may helix (Fig. 1 A and B, red), our target protein would bind to the hamper 3D reconstruction. Despite the numerous successes of approximate center of our support structure. By designing ssDNA single-particle analysis in recent years, preparing suitable cryo- overhangs (T10) on both the top and bottom faces of the pillar EM samples therefore remains a nontrivial task. Often, estab- (Fig. 1 A–C, blue), we aimed to expedite a preferred orientation of lishing optimal ice thickness and particle distribution in the ice the support in the ice layer.