Grafix Sample Preparation For Single-particle Electron Cryomicroscopy
Heinz Francis
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We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.
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We thank H. Kohansal, T. Conrad and W. Jahn for expert technical assistance, U. Gringer and M. Brecht for preparing the RNA editing complex, M. Rodnina and K. Gromadski for preparing the 70S ribosome complex, and C. Will for preparing the 17S U2 snRNP. The work was supported by grants from the Federal Ministry of Education and Research, Germany and from the Sixth Framework Programme of the European Union via 3DRepertoire (to H.S.). Work in the laboratory of J.M.P. is supported by Boehringer Ingelheim and by Spots of Excellence of the city of Vienna. N.F. is supported by a Boehringer-Ingelheim fellowship. E.W. is supported by a 'Studienstiftung des deutschen Volkes' fellowship.
Figure 1. Generic protein purification workflow and different membrane protein stabilization strategies using artificial membranes. (A) Cytoplasmic or membrane proteins are initially expressed in liquid or solid cultures, and pellets are stored after harvesting by centrifugation. Different physical or chemical cell disruption methods are utilized for releasing cytoplasmic proteins into solution or to obtain cell membrane extracts. Impure cytoplasmic proteins or solubilized cell membranes containing the protein of interest are purified by combination of different fast protein liquid chromatography (FPLC) methods. After protein stability, integrity and activity is verified by various biophysical techniques. The final sample concentration and buffer composition are adjusted before EM grid preparation. (B) Protein transmembrane domains are protected by the hydrophobic cell membrane phospholipid acyl chains. Micelles are spherical vesicles in which the detergent hydrophobic chains face inward and the hydrophilic polar heads face outward. Bicelles are obtained by a mixture of lipids and short chain detergents. The lipids will interact with the protein to form a lipid bilayer and the detergent will form the rim of the bicelle. Micelles will form after the solubilization of the membrane protein by detergents. SMALP (styrene-maleic acid lipid particles) are polymeric nanoparticles that protect the acyl chain of the lipid bilayer. Nanodiscs are lipid bilayers stabilized by wrapping a belt of amphipathic helix-rich membrane scaffold proteins (MSPs) around the detergent-solubilized membrane proteins. Amphipol polymers wrap around the hydrophobic patches of the membrane protein to form a stable complex in solution. Liposomes are artificial spherical lipid membranes where membrane proteins can assemble.
Figure 2. Different designs of a TEM (transmission electron microscopy) grid and semi-automated method for specimen vitrification. (A) Examples of a TEM grid with irregular hole size foil (Lacey) or with defined hole diameter and spacing (Quantifoil). (B) An automated plunge-freezing device is commonly used for specimen vitrification. Sample is applied with a pipette at the surface of the cryo-EM grid and sample excess is removed by blotting with filter paper, followed by immediate freezing in liquid ethane. The specimen can be frozen on a grid with (i) or without (ii) a thin continuous film made of different materials. TEM grids with different grid mesh, foil and grid support materials can be used during specimen freezing.
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Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM). In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes. Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging. Here, we describe strategies for the purification and stabilization of Pol I complexes, exemplarily considering advantages and disadvantages of the methodology. We further provide an easy-to-implement protocol for the coating of EM-grids with self-made carbon support films. In sum, we present an efficient workflow for cryo-grid preparation and optimization, including early stage cryo-EM screening that can be adapted to a wide range of soluble samples for high-resolution structure determination.
The success of structure determination by single-particle cryo-EM depends on high-quality biochemical preparation and characterization of the molecule(s) of interest. Buffer optimization and stabilization of complexes should be done in the initial phase of a project, strategies are described in detail [20]. We previously presented protocols for the purification of 14-subunit, 590 kDa Pol I complexes and their characterization in vitro [21, 22]. Starting from this, we detail the specimen features which are important for successful structure determination using single-particle cryo-EM and suggest approaches for their optimization. For this purpose, we compare complex preparation- and assembly strategies using endogenously purified Pol I and recombinant transcription factors on nucleic acid templates. Transcription factor complexes can be (a) assembled on a biotinylated DNA, enriched using the interaction with bead-coupled streptavidin and eluted with restriction enzymes [23]. Alternatively (b), size exclusion chromatography (SEC) may yield homogenous and stable complexes that are well-suited for cryo-grid preparation. Large macromolecules can also be enriched using density gradient centrifugation protocols (c). As such, the gradient-fixation (GraFix)-method relies on a sedimentation step coupled with an intra- and intermolecular cross-linking step combining purification and complex stabilization [24, 25]. Following GraFix, a buffer exchange is required to remove sucrose or glycerol that would otherwise interfere with the subsequent freezing process and may increase background noise in cryo-EM images. Generally, cross-linking of protein-protein or protein-nucleic acid complexes can improve their stability during the grid preparation process. This cross-linking can be coupled to a purification step (as in GraFix), or performed directly before grid-plunging (d). These preparation techniques can also be combined, for example, carrying out an SEC run after batch cross-linking. Such a strategy combines the advantages of sample stabilization and purification while directly including the transition to a suitable buffer system but requires larger quantities of sample.
In addition to sample optimization and the choice of grid type, various physical parameters influence the preparation of cryo-EM grids. Glow discharge settings, grid type variation, buffer choice, and the mechanics of the blotting device should be carefully considered for each individual sample.
In general, we recommend initial optimization using negative staining EM, followed by two stages of cryo-EM screening (Fig. 1). The first cryo-screening stage aims at an evaluation of grid types, support films, or blotting conditions and gives information on sample behavior in ice. In a second phase of cryo-screening, intermediate-resolution single-particle maps may be reconstructed. Phase I cryo-screening results yield insights into sample behavior, whereas results of the second cryo-screening phase indicate whether the sample is suitable for high-resolution data collection by identifying flexibilities within the macromolecular complex and bias in orientation distribution.
Construct a floatation chamber well in advance (compare Fig. 2). The design was described in [33] and should be adaptable in a scientific workshop. A commercial version (SKU 10840) of a similar apparatus is available from LADD Research Industries (Williston, VT, USA). We also use this flotation chamber to transfer surface assembled graphene oxide [44] onto EM grids.
Place metal support mesh on beams in floatation chamber and place marked filter paper on top of metal mesh. Close the flexible outlet tube at the chamber bottom using a clip or connect to peristaltic pump.
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