Supporting Information

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1 Supporting Information Battisti et al /pnas SI Text SI Materials and Methods. Virus propagation and purification. Egggrown Newcastle disease virus (NDV; strain B1), harvested at 42 h postinfection, was purified as previously described (1). For cryoelectron tomography the virus was placed into a TNE buffer (0.02 M Tris, 0.12 M NaCl, and M EDTA, ph 8.0). The total protein concentration was adjusted to 1 3 mg ml (corresponding to a virus titer of to pfu ml). SDS-PAGE was used to detect the presence of viral proteins and assess purity. Cryoelectron tomography and 3D reconstruction. A suspension of BSA-conjugated 10 nm gold beads (BSA Gold Tracers; Aurion) was added to the purified NDV suspension. Next, 3.5 μl aliquots of the virus gold mixture were applied to holey carbon covered EM grids (Quantifoil Micro Tools GmbH), blotted with filter paper, and plunged into liquid ethane. The vitrified samples were imaged using a Titan Krios (FEI) field emission gun microscope operated at 300 kv. Additionally, the microscope was equipped with an energy filter (Gatan, Inc.) operated in zero-energy-loss mode with a slit width of ev. Images were recorded on a 2,048- by 2,048-pixel CCD camera at a magnification of 19;500 (0.75 nm separation between pixels). The tilt-series images were obtained over an angular range of 69 in 1.5 increments at a defocus of 8 9 μm. The cumulative electron dose for the complete series was in the range of electron Å 2. The FEI Batch Tomography software was used to perform automatically tilting, tracking, focusing, and image acquisition. Tiltseries images were aligned using the colloidal gold particles as fiducial markers and reconstructed using the weighted back-projection method as implemented in the IMOD software package (2). The resolution of tomograms is difficult to quantify and is exceedingly isotropic because of the missing wedge of data. However, the best resolution in the direction perpendicular to the electron beam is better than 7 nm, based on the ability to observe the spacing between matrix protein dimers. Alignment and averaging of matrix protein subunits. In order to improve the matrix protein density, adjoining regions of the matrix protein layer plus associated membrane were aligned and averaged using the AVE and IMP programs from the Uppsala Software Factory (3, 4). The best rotational and translational parameters relating each region to its neighbor were determined by maximizing the correlation coefficient between neighboring regions. Using a density threshold that would make the averaged membrane density about 4 nm thick (corresponding to 1.7 standard deviations above the mean density value), the volume of one subunit was measured to be about 85 nm 3 using the Chimera visualization tool (5). Assuming an average protein density of 1.35 g cm 3, the molecular mass of one subunit would be about 70 kda, consistent with each subunit being a dimer of 76 kda. Cloning, expression, and purification of newcastle disease virus matrix protein. The full-length gene of the NDV matrix protein (6) was amplified by PCR and ligated into the pet28a (Merck KGaA) expression vector, which has an N-terminal 6-histidine tag sequence. The expression plasmid was transfected into Escherichia coli, strain BL21 (Rosetta), for protein expression. The cells were grown at 37 C until the optical density reached , at which point 1 M IPTG was added to induce the expression of the protein. This culture was then grown at 18 C for 20 h, harvested by centrifugation, and suspended in Hepes buffer (500 mm NaCl, 50 mm Hepes, ph 8.0). The harvested cells were lysed and centrifuged at 15;000 g for 30 min. The supernatant was applied to a nickel affinity column and washed in an imidazole gradient. The fraction containing the matrix protein was concentrated by centrifugation and further purified by size exclusion chromatography using a S200 Superdex column (GE Healthcare). The purified matrix protein was concentrated to 3 mg ml and frozen in liquid nitrogen for further use. Crystallization, X-ray data collection, and structure determination of the matrix protein. Crystallization conditions were screened by the sitting drop vapor diffusion method using a Honeybee crystallization robot (Digilab, Inc.). Almost 500 conditions were screened using Hampton (Hampton Research) and Emerald (Emerald Biosystems) crystallization kits. Each 1 μl drop was a mixture of 0.5 μl protein and 0.5 μl well solution. Small, 0.01 mm diameter plate-like crystals were obtained initially in several conditions. These conditions were further optimized to improve the size and quality of the crystals, which eventually could be grown to about 0.3 mm in 1 week. The crystals were mounted on loops and flash frozen in liquid nitrogen after soaking in the cryoprotectant, which consisted of a mixture of the mother liquor with 25% (vol vol) glycerol. Mercury-derivatized crystals were obtained by cocrystallization with various mercury compounds. Two forms of native crystals and one form of a nonisomorphous Hg-derivative crystal were found. Diffraction data were collected at the Advanced Photon Source beamline 23 ID-B/D using crystals maintained at 100 K and a MAR 300 CCD detector (Table S1). All data were processed using the HKL2000 program (7). The Matthews coefficient for the Hg derivative was 2.4 Å 3 Da assuming two molecules in the asymmetric unit. Phases were determined with the Phenix program (8) using single-wavelength anomalous dispersion data. Twelve heavy-atom sites were found in the crystallographic asymmetric unit. The initial phases were iteratively refined using density modifications. The programs Phenix and Resolve (9) were used to build the structure into the electron density map. This structure was then refined using the program Refmac (10) in CCP4 (11) and viewed with the program Coot (12). Molecular replacement was used to determine the structure of the two native datasets using the program Molrep (13) in CCP4 (11). The final R working and R free factors for the three datasets were better than 24.5% and 28.8%, respectively (Table S1). The rmsd between noncrystallographic symmetry (NCS)-equivalent Cα atoms within each structure was 0.2 Å and the NCS rotation was within 0.3 of 180. The two domains within each monomer are related by a 67 rotation, which is coincident with the twofold NCS axis. The NDV matrix protein structure was visualized using PyMol (Schrödinger, LLC) and represented schematically using Top- Draw (14). A structure-based sequence alignment comparing the NDV matrix protein to other Mononegavirales matrix proteins was carried out using the program HOMOlogy (15). The crystallographic matrix dimer was fitted into the tomographic matrix density using the program EMfit (16). Helical reconstruction of the nucleocapsid. An averaged one-dimensional Fourier transform from 25 nucleocapsid regions in the NDV tomograms, scanned along their lengths, had one large coefficient corresponding to a 7 nm repeat. The nucleocapsid density was helically averaged assuming different pitch and twist values using a script implemented in the EMAN package (17). Plotting the correlation coefficient between the averaged and unaveraged nucleocapsid maps as a function of twist and pitch 1of8

2 resulted in a line of high correlation with a positive slope, indicative of a right-handed helix with a pitch of 7 nm. The line of high-correlation values was fairly continuous, indicating that the tomograms were not of sufficient resolution to discern independent nucleocapsid subunits. Combining the averaged matrix protein tomographic density with the averaged tomographic nucleocapsid structures. A 60 nm long averaged helical nucleocapsid structure was fitted into 25 nearly straight nucleocapsid densities from six different tomograms using the program IMP (3). Similarly, the averaged matrix protein region was fitted into the matrix densities neighboring the fitted nucleocapsids. For 18 of the 25 nucleocapsid matrix interfaces the 7 nm pitch of the nucleocapsid was found to be in register (10 ) with the distance between matrix protein subunits along the array diagonal. 1. McGinnes LW, Pantua H, Reitter J, Morrison TG (2006) Newcastle disease virus: Propagation, quantification, and storage. Curr Protoc Microbiol Chapter 15:Unit 15F Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of threedimensional image data using IMOD. J Struct Biol 116: Jones TA (1992) A set of averaging programs. Molecular Replacement, eds Dodson EJ, Gover S, Wolf W (SERC Daresbury Laboratory, Warrington, UK), pp Kleywegt GJ, Jones TA (1994) Halloween masks and bones. From First Map to Final Model, eds Bailey S, Hubbard R, Waller D (SERC Daresbury Laboratory, Warrington, UK), pp Pettersen EF, et al. (2004) UCSF Chimera: A visualization system for exploratory research and analysis. J Comput Chem 25: McGinnes LW, Morrison TG (1987) The nucleotide sequence of the gene encoding the Newcastle disease virus membrane protein and comparisons of membrane protein sequences. Virology 156: Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol, ed Carter CW, Jr (Academic Press, New York), Vol 276, pp Adams PD, et al. (2002) PHENIX: Building new software for automated crystallographic structure determination. Acta Crystallogr Sect D: Biol Crystallogr 58: Terwilliger TC (2003) Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr Sect D: Biol Crystallogr 59: Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr Sect D: Biol Crystallogr 53: Winn MD, et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr Sect D: Biol Crystallogr 67: Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr Sect D: Biol Crystallogr 60: Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular replacement. J Appl Crystallogr 30: Bond CS (2003) TopDraw: A sketchpad for protein structure topology cartoons. Bioinformatics 19: Rao ST, Rossmann MG (1973) Comparison of super-secondary structures in proteins. J Mol Biol 76: Rossmann MG, Bernal R, Pletnev SV (2001) Combining electron microscopic with x-ray crystallographic structures. J Struct Biol 136: Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: Semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128: Fig. S1. The NDV nucleocapsid. (A) A repeating pattern along the length of NDV nucleocapsids was seen in the tomographic data. The image was generated by averaging 20 layers of voxels over a thickness of 30 nm. Black represents high density. (B) The Fourier transform of a one-dimensional scan over the length of the image has a large peak corresponding to a helical pitch of 7 nm. (C) Reconstruction of the helical nucleocapsid assuming a pitch of 7 nm and a twist of 18 subunits/turn. 2of8

3 Fig. S2. Nonreducing, silver-stained SDS-PAGE of purifiedvirus. The center and right lanes were loaded with 1and 2.5μL of purified NDV, respectively. The left lane was loaded with a molecular weight marker. The viral proteins and marker molecular weights are identified in the figure. Although the majority of virions do not have an ordered matrix protein layer, the matrix protein is present at about the same relative abundance as the other structural proteins in the virions. 3of8

4 Fig. S3. The relationship between the matrix layer and the nucleocapsid filaments. (A and B) Tomographic sections showing portions of the nucleocapsid of two virions (Top). The yellow arrowsindicateportionsofthe nucleocapsid that are roughlyinregister with theadjoiningmatrix array (Middle). The relationship between the nucleocapsids and matrix array are shown schematically (Bottom). The yellow rectangles represent portions of the nucleocapsid and the blue squares indicate the approximate orientation of the matrix array, the diagonal of which forms a roughly 45 angle with the long axis of elongated virions. High density is black and the sections represent an average of five planes of voxels (a thickness of approximately 75 nm). The scale bars represent 25 nm. (C) The averaged helical nucleocapsid structure (yellow density) was fitted into the virion depicted in A. A number of copies of the averaged matrix protein subunit (blue density) have been placed into a portion of the matrix array shown in A. The rotational and translational elements determined during the averaging procedure were used to place the matrix density. The 7 nm repeating units of the matrix array and nucleocapsid structure are oriented in roughly the same direction. The scale bar represents 25 nm. (D) The structures depicted in C, rotated approximately 55 about the horizontal axis. The scale bar represents 25 nm. (E) The matrix protein dimer structure (blue ribbon) and averaged helical nucleocapsid structure (yellow density) were fitted into the virion depictedinb. The 7 nm repeating units of the matrix array and nucleocapsid structure are oriented in roughly the same direction. (F) The structures depicted in E, rotated approximately 90 about the horizontal axis. The scale bar represents 25 nm. 4of8

5 Fig. S4. Gel filtration indicates the matrix protein is dimeric. (Left) The matrix protein was purified using a Superdex /60 GL (GE Healthcare) sizeexclusion column. The arrows indicate three different elution peaks for NDV matrix protein. The dashed vertical lines indicate the elution peaks of four molecular weight standards. A comparison between the matrix protein elution positions and the molecular weight standards indicates that the matrix protein is mainly dimeric or forming higher-order oligomers in solution, though some monomeric protein is present. (Inset, Right) SDS-PAGE of the protein eluted from the monomeric (P3), dimeric (P2), and higher-order oligomer (P1) peaks. The left lane was loaded with a molecular weight marker. Marker molecular weights are indicated in the figure. 5of8

6 Fig. S5. Conservation of residues in the matrix protein dimer interface. (A) A surface representation of the NDV matrix protein monomer. The monomer monomer interface within a dimer is outlined by a thick black line. The dark blue residues are completely conserved among 10 members of the Paramyxovirinae subfamily. Lighter blue indicates partial conservation and white indicates no significant conservation. Of residues in the dimer interface, 36.6% (15 out of 41) are completely conserved, whereas only 14.3% of other surface residues (34 out of 237) are completely conserved. (B) The residues on the opposite side of the monomer to the dimer interface are less conserved among paramyxoviruses. 6of8

7 Fig. S6. Structure-basedsequencealignment ofmononegavirales matrix proteins. The crystal structures of the respiratory syncytial virus matrix protein, Borna virus matrix protein, and Ebola virus VP40 were structurally aligned with the NDV matrix protein. Secondary structural elements for NDV are shown above the sequence alignment. Residues that are conserved for at least two of the four aligned proteins are highlighted in red. 7of8

8 Table S1. Crystallographic data collection and refinement statistics* Native 1 Native 2 Hg derivative Data collection X-ray source 23-ID-D 23-ID-D 23-ID-B Wavelength (Å) Cell dimensions a ¼ 163, b ¼ 47, c ¼ 117; β ¼ 131, α ¼ γ ¼ 90 a ¼ 247, b ¼ 45, c ¼ 58; β ¼ 103, α ¼ γ ¼ 90 a ¼ 46.8, b ¼ c ¼ 141.8;α ¼ β ¼ γ ¼ 90 Space group C2 C2 P22121 No. monomers per asymmetric unit Oscillation angle No. frames Resolution range R merge 0.088(0.612) 0.078(0.602) 0.116(0.364) Completeness 99.6(100) 93.9(94.6) 100(100) Redundancy Refinement Resolution (Å) R working (%) R free (%) Average B factor (Å 2 ) Rmsd bonds (Å) Rmsd angles ( ) Ramachandran disallowed *Values in parentheses throughout the table correspond to the last shell. R merge ¼ ΣjI hiij ΣI, where I is the measured intensity for reflections with indices hkl. R working ¼ ΣjjF obs j jf calc jj ΣjF obs j. R free has the same formula as R working, except that the calculation was made with the structure factors from the test set. Table S2. Fitting of the crystallographic matrix protein dimer into the averaged matrix protein density from cryoelectron tomography using the EMfit program Sumf* Clash -Den % 8.7% *Sumf is the mean density height averaged over all atoms where the maximum density in the electron density map is set to 100. Clash represents the percentage of atoms in the model that approach closer than 5 Å to neighboring matrix dimers. The percentage of atoms in density less than the mean density value is given by Den. Table S3. Sequence and structural comparisons between the 360 Cα atoms of the NDV matrix protein and other Mononegavirales* matrix proteins Molecule Dali score Rmsd between Cα atoms No. of aligned Cα atoms Total no. of Cα atoms PDB ID Sequence identity RSV M VQP 7.5% BDV M F1J 7.8% EBV M ES6 4.6% M, matrix protein; PDB, Protein Data Bank; RSV, respiratory syncytial virus; BDV, Borna disease virus: EBV, Ebola virus. *The structure of the vesicular stomatitis virus matrix protein was not included because it has a different fold. Alignment of the BDV matrix protein with the C-terminal domain of NDV M. Alignment of the Ebola virus matrix protein with the N-terminal domain of NDV M. 8of8