The images in each stack were uniformly masked to include information with an ... software package (Apple, Inc.) and the movies were compiled to show images ...
Electronic Supplementary Information (ESI) Experimental Methods DLP preparation. Simian rotavirus (strain SA11-‐4F) DLPs were purified as previously described11. Transcription reactions (25-‐μl each) were in carried out in eppendorf tubes incubated for ~30 minutes at 37°C.6 Briefly, each mixture contained the following: 1 μg DLPs prepared in 100 mM Tris-‐HCl pH 7.5, 6 mM MgAc, 4 mM DTT, 2 mM each of ATP, GTP, CTP, UTP, and 1μl RNasin (Promega Corp., Madison, WI). Following the 30-‐minute incubation period, 3-‐μl aliquots of the reaction mixtures were applied to antibody-‐ decorated SiN chips used for subsequent experiments. Antibody-‐tethering procedures for DLPs. SiN microchips containing integrated microwells (Protochips, Inc.) were coated with Nickel-‐nitrilotriacetic acid (Ni-‐NTA) lipid monolayers as previously described.10 The Ni-‐NTA lipid coatings were comprised of 25% Ni-‐NTA lipids and 75% 1,2-‐dilauryl-‐phosphatidylcholine (DLPC) filler lipids (Avanti Polar Lipids). Adaptor proteins were added sequentially to the Ni-‐NTA coated microchips and included His-‐tagged Protein A (3-‐μl aliquots of 0.01 mg ml-‐1; Abcam) in buffer solution containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2 and 10 mM CaCl2. The protein A aliquots were incubated for 1 minute on each microchip prior to removing the excess solution by blotting with filter paper. Next, we added VP6-‐specific guinea pig polyclonal antisera (#53963) (3-‐μl aliquots of 0.01 mg ml-‐1) prepared in 50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2 and 10 mM CaCl2 buffer solution. Following a 1-‐minute incubation step, the excess solution was removed using a Hamilton syringe. Enzymatically active DLPs (2-‐μl aliquots of 0.1 mg ml-‐1) were added to the antibody-‐decorated microchips
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for a 2-‐minute incubation. Microchips containing the tethered DLPs were then loaded into the Poseidon 200 liquid specimen holder (Protochips, Inc.) as described below. Antibody-‐ tethered grids for cryo-‐EM control experiments were performed using the same procedures, but using holey carbon grids (C-‐flat -‐ 2/1 grids; Protochips, Inc.) rather than microchips. Frozen-‐hydrated specimens were prepared by plunge-‐freezing the antibody-‐ tethered DLPs into a liquid ethane slurry using a Gatan Cryoplunge™ 3 equipped with GentleBlot capabilities (Gatan, Inc.) and employing a one-‐sided blotting step for approximately 8 seconds. Electron Microscopy. All specimens were examined using a FEI Spirit BioTwin TEM (FEI Company, Hillsboro, OR, USA) equipped with a LaB6 filament operating at 120kV under low-‐dose conditions (< 1 electron per Å2). Images were recorded using a FEI Eagle 2k HS CCD camera having a pixel size of 30 μm. Images of DLPs in liquid and in ice were recorded at a nominal magnification of 60,000 × with a final sampling of ~5 Å per pixel using a defocus range of -‐1.5 to -‐3 μm. For image series acquisition, we collected sequential images at intervals ranging from 0.25 – 1 s-‐1. For the image series analyzed here, we selected representative DLPs from the images using the PARTICLE software package (http://www.image-‐analysis.net/EM/). For cryo-‐EM imaging, we employed the same TEM and imaging parameters. Cinematography. Intact particles sufficiently distanced from each other were boxed out to create an image stack through the acquired image series using the PARTICLE software package. The images in each stack were uniformly masked to include information with an
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80-‐nm diameter then colorized for visualization purposes and movie production (Movies S1 – S3). Images within each stack were also subjected to a density threshold using a significance level cutoff of 3σ as described in other work.12, 15 Contour maps of the remaining density were compiled for movie production (Movies S4 – S6) and quantitative analysis. Images sequences and contour maps were imported into the iMovie 10.0.7 software package (Apple, Inc.) and the movies were compiled to show images cycling at 0.5-‐second intervals. The movies were exported .mov format. 3D reconstructions. Individual DLPs were selected from cryo-‐EM images using the PARTICLE software package utilizing a box size of 120 nm. The selected particles were output as MRC image stacks and imported into the RELION software package13 for 3D reconstruction calculations. Within the RELION package we used refinement parameters included a pixel size of 5 Å, a reference model low-‐pass filtered to 50 Å, and a regularization parameter of T= 4. We enforced icosahedral symmetry over an angular search space of 7.5° while implementing refinement procedures for 25 cycles outputting the reconstructions in Figure 3a and 3b with a resolution of ~2.8 and ~2.5 nm, respectively. Slices through each of the reconstructions revealed internal densities of the particles. The slices were taken at ~20 nm intervals ending at the midsection of each structure.
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Fig. S1. (a) Purified rotavirus DLPs characterized by SDS-‐PAGE and silver stain analysis indicate the presence of viral proteins (VP1, VP2, VP3, and VP6). Purified DLPs were enzymatically activated upon the addition of nucleotides, including ATP, to produce [32P]-‐ labeled mRNA transcripts. Reaction mixtures lacking ATP fail to produce appreciable levels of mRNA transcripts. (b) Schematic to indicate the immunocapture procedure used to tether asynchronously transcribing DLPs to antibody (IgG)-‐decorated surfaces via protein A adaptors. (c) Transcribing DLPs tethered to EM grids in the presence of VP6-‐specific IgGs show varying lengths of associated mRNA transcripts (white arrows) in cryo-‐EM images. (d) DLPs prepared in the absence of nucleotides needed for transcription do not show associated mRNA in cryo-‐EM images. (e) EM specimens prepared in the absence of IgGs generally failed to recruit DLPs. Scale bar is 100 nm. Information in panels (a) and (b) are adapted from previous work.6
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Fig. S2. Representative particle images of DLPs contained in liquid were selected from the image series recorded over 10 seconds. The selected particles were then contrast-‐inverted, and colorized for visualization purposes. Scale bar is 30 nm. Please see associated Movies S1 – S3.
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Movie S1. Time-‐resolved movie of a rotavirus DLP in liquid. Representative particle images of DLPs in liquid were uniformly masked and colorized blue to include information with an 80-‐nm diameter. Image sequences were imported into the iMovie 10.0.7 software package (Apple, Inc.) and the movie was compiled to show images cycling at 0.5 s intervals. The movie was looped for a uniform time of 15 s and exported using .mov format. Movie S2. Time-‐resolved movie of a rotavirus DLP in liquid. Representative particle images of DLPs in liquid were uniformly masked and colorized gray to include information with an 80-‐nm diameter. Image sequences were imported into the iMovie 10.0.7 software package (Apple, Inc.) and the movie was compiled to show images cycling at 0.5 s intervals. The movie was looped for a uniform time of 15 s and exported using .mov format. Movie S3. Time-‐resolved movie of a rotavirus DLP in liquid. Representative particle images of DLPs in liquid were uniformly masked and colorized green to include information with an 80-‐nm diameter. Image sequences were imported into the iMovie 10.0.7 software package (Apple, Inc.) and the movie was compiled to show images cycling at 0.5 s intervals. The movie was looped for a uniform time of 15 s and exported using .mov format. Movie S4. Movie to indicate the mobile units of Particle 1 (blue) in liquid. Representative particle images of DLPs in liquid were selected (zoomed in) and subjected to a density threshold filter using a significance level cutoff of 3σ. Contour maps of the resulting particles components composed of genomic RNA and associated proteins were
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uniformly masked and imported as image sequences into the iMovie 10.0.7 software package (Apple, Inc.). The movie was compiled to show images cycling at 0.5 s intervals and exported using .mov format. Additional quantitative analysis of these images (Fig. 2b) revealed that this particle series exhibited the greatest number of pixel displacements in comparison to the other analyzed particles. Movie S5. Movie to indicate the mobile units of Particle 2 (gray) in liquid. Representative particle images of DLPs in liquid were selected (zoomed in) and subjected to a density threshold filter using a significance level cutoff of 3σ. Contour maps of the resulting particles components composed of genomic RNA and associated proteins were uniformly masked and imported as image sequences into the iMovie 10.0.7 software package (Apple, Inc.). The movie was compiled to show images cycling at 0.5 s intervals and exported using .mov format. Additional quantitative analysis of these images (Fig. 2b) revealed that this particle series exhibited the fewest pixel displacements in comparison to the other analyzed particles. Movie S6. Movie to indicate the mobile units of Particle 3 (green) in liquid. Representative particle images of DLPs in liquid were selected (zoomed in) and subjected to a density threshold filter using a significance level cutoff of 3σ. Contour maps of the resulting internal particles components, primarily composed of genomic RNA and associated proteins, were uniformly masked and imported as image sequences into the iMovie 10.0.7 software package (Apple, Inc.). The movie was compiled to show images cycling at 0.5 s intervals and exported using .mov format. Additional quantitative analysis
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of these images (Fig. 2b) revealed that this particle series exhibited an intermediate number of pixel displacements in comparison to the other analyzed particles.
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