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Supporting InformationBattisti et al. 10.1073/pnas.1210275109SI TextSI Materials and Methods. Virus propagation and purification. Egg-grown Newcastle disease virus (NDV; strain B1), harvested at42 h postinfection, was purified as previously described (1). Forcryoelectron tomography the virus was placed into a TNE buffer(0.02 M Tris, 0.12 M NaCl, and 0.001 M EDTA, pH 8.0). The totalprotein concentration was adjusted to 1–3 mg∕mL (correspondingto a virus titer of 1010 to 1011 pfu∕mL). SDS-PAGE was used todetect the presence of viral proteins and assess purity.

Cryoelectron tomography and 3D reconstruction. A suspension ofBSA-conjugated 10 nm gold beads (BSA Gold Tracers; Aurion)was added to the purified NDV suspension. Next, 3.5 μL aliquotsof the virus–gold mixture were applied to holey carbon–coveredEM grids (Quantifoil Micro Tools GmbH), blotted with filterpaper, and plunged into liquid ethane. The vitrified samples wereimaged using a Titan Krios (FEI) field emission gun microscopeoperated at 300 kV. Additionally, the microscope was equippedwith an energy filter (Gatan, Inc.) operated in zero-energy-lossmode with a slit width of 20–30 eV. Images were recorded ona 2,048- by 2,048-pixel CCD camera at a magnification of19;500 × (0.75 nm separation between pixels). The tilt-seriesimages were obtained over an angular range of �69° in 1.5° in-crements at a defocus of 8–9 μm. The cumulative electron dosefor the complete series was in the range of 150–200 electron∕Å2.The FEI Batch Tomography software was used to perform auto-matically tilting, tracking, focusing, and image acquisition. Tilt-series images were aligned using the colloidal gold particles asfiducial markers and reconstructed using the weighted back-pro-jection method as implemented in the IMOD software package(2). The resolution of tomograms is difficult to quantify and isexceedingly isotropic because of the missing wedge of data. How-ever, the best resolution in the direction perpendicular to theelectron beam is better than 7 nm, based on the ability to observethe spacing between matrix protein dimers.

Alignment and averaging of matrix protein subunits. In order to im-prove the matrix protein density, adjoining regions of the matrixprotein layer plus associated membrane were aligned and aver-aged using the AVE and IMP programs from the Uppsala Soft-ware Factory (3, 4). The best rotational and translationalparameters relating each region to its neighbor were determinedby maximizing the correlation coefficient between neighboringregions. Using a density threshold that would make the averagedmembrane density about 4 nm thick (corresponding to 1.7 stan-dard deviations above the mean density value), the volume of onesubunit was measured to be about 85 nm3 using the Chimeravisualization tool (5). Assuming an average protein density of1.35 g∕cm3, the molecular mass of one subunit would be about70 kDa, consistent with each subunit being a dimer of 76 kDa.

Cloning, expression, and purification of newcastle disease virus matrixprotein. The full-length gene of the NDV matrix protein (6) wasamplified by PCR and ligated into the pET28a (Merck KGaA)expression vector, which has an N-terminal 6-histidine tag se-quence. The expression plasmid was transfected into Escherichiacoli, strain BL21 (Rosetta), for protein expression. The cells weregrown at 37 °C until the optical density reached 0.6–0.8, at whichpoint 1 M IPTG was added to induce the expression of the pro-tein. This culture was then grown at 18 °C for 20 h, harvested bycentrifugation, and suspended in Hepes buffer (500 mM NaCl,50 mM Hepes, pH 8.0). The harvested cells were lysed and cen-

trifuged at 15;000 × g for 30 min. The supernatant was appliedto a nickel affinity column and washed in an imidazole gradient.The fraction containing the matrix protein was concentrated bycentrifugation and further purified by size exclusion chromato-graphy using a S200 Superdex column (GEHealthcare). The pur-ified matrix protein was concentrated to 3 mg∕mL and frozen inliquid nitrogen for further use.

Crystallization, X-ray data collection, and structure determination ofthe matrix protein.Crystallization conditions were screened by thesitting drop vapor diffusion method using a Honeybee crystalliza-tion robot (Digilab, Inc.). Almost 500 conditions were screenedusing Hampton (Hampton Research) and Emerald (EmeraldBiosystems) crystallization kits. Each 1 μL drop was a mixtureof 0.5 μL protein and 0.5 μL well solution. Small, 0.01 mm dia-meter plate-like crystals were obtained initially in several condi-tions. These conditions were further optimized to improve thesize and quality of the crystals, which eventually could be grownto about 0.3 mm in 1 week. The crystals were mounted on loopsand flash frozen in liquid nitrogen after soaking in the cryoprotec-tant, which consisted of a mixture of the mother liquor with 25%(vol∕vol) glycerol. Mercury-derivatized crystals were obtained bycocrystallization with various mercury compounds. Two forms ofnative crystals and one form of a nonisomorphous Hg-derivativecrystal were found. Diffraction data were collected at the Ad-vanced Photon Source beamline 23 ID-B/D using crystals main-tained at 100 K and a MAR 300 CCD detector (Table S1). Alldata were processed using the HKL2000 program (7).

TheMatthews coefficient for the Hg derivative was 2.4 Å3∕Daassuming two molecules in the asymmetric unit. Phases were de-termined with the Phenix program (8) using single-wavelengthanomalous dispersion data. Twelve heavy-atom sites were foundin the crystallographic asymmetric unit. The initial phases wereiteratively refined using density modifications. The programsPhenix and Resolve (9) were used to build the structure into theelectron density map. This structure was then refined using theprogram Refmac (10) in CCP4 (11) and viewed with the programCoot (12). Molecular replacement was used to determine thestructure of the two native datasets using the program Molrep(13) in CCP4 (11). The final Rworking and Rfree factors for thethree 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 andthe NCS rotation was within 0.3° of 180°. The two domains withineach monomer are related by a 67° rotation, which is coincidentwith 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 theNDV matrix protein to other Mononegavirales matrix proteinswas carried out using the program HOMOlogy (15). The crystal-lographic matrix dimer was fitted into the tomographic matrixdensity using the program EMfit (16).

Helical reconstruction of the nucleocapsid.An averaged one-dimen-sional Fourier transform from 25 nucleocapsid regions in theNDV tomograms, scanned along their lengths, had one largecoefficient corresponding to a 7 nm repeat. The nucleocapsiddensity was helically averaged assuming different pitch and twistvalues using a script implemented in the EMAN package (17).Plotting the correlation coefficient between the averaged andunaveraged nucleocapsid maps as a function of twist and pitch

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resulted in a line of high correlation with a positive slope, indi-cative of a right-handed helix with a pitch of 7 nm. The line ofhigh-correlation values was fairly continuous, indicating thatthe tomograms were not of sufficient resolution to discern inde-pendent nucleocapsid subunits.

Combining the averaged matrix protein tomographic density with theaveraged tomographic nucleocapsid structures. A 60 nm–long aver-

aged helical nucleocapsid structure was fitted into 25 nearlystraight nucleocapsid densities from six different tomogramsusing the program IMP (3). Similarly, the averaged matrix proteinregion was fitted into the matrix densities neighboring the fittednucleocapsids. For 18 of the 25 nucleocapsid–matrix interfacesthe 7 nm pitch of the nucleocapsid was found to be in register(�10°) with the distance between matrix protein subunits alongthe array diagonal.

1. McGinnes LW, Pantua H, Reitter J, Morrison TG (2006) Newcastle disease virus: Propa-gation, quantification, and storage. Curr Protoc Microbiol Chapter 15:Unit 15F.2.

2. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76.

3. Jones TA (1992) A set of averaging programs.Molecular Replacement, eds Dodson EJ,Gover S, Wolf W (SERC Daresbury Laboratory, Warrington, UK), pp 91–105.

4. Kleywegt GJ, Jones TA (1994) Halloween…masks and bones. From First Map to FinalModel, eds Bailey S, Hubbard R, Waller D (SERC Daresbury Laboratory, Warrington,UK), pp 59–66.

5. Pettersen EF, et al. (2004) UCSF Chimera: A visualization system for exploratory re-search and analysis. J Comput Chem 25:1605–1612.

6. McGinnes LW, Morrison TG (1987) The nucleotide sequence of the gene encoding theNewcastle disease virus membrane protein and comparisons of membrane proteinsequences. Virology 156:221–228.

7. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscilla-tion mode. Methods Enzymol, ed Carter CW, Jr (Academic Press, New York), Vol 276,pp 307–326.

8. Adams PD, et al. (2002) PHENIX: Building new software for automated crystallographicstructure determination. Acta Crystallogr Sect D: Biol Crystallogr 58:1948–1954.

9. Terwilliger TC (2003) Automated main-chain model building by template matchingand iterative fragment extension. Acta Crystallogr Sect D: Biol Crystallogr 59:38–44.

10. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structuresby the maximum-likelihood method. Acta Crystallogr Sect D: Biol Crystallogr53:240–255.

11. Winn MD, et al. (2011) Overview of the CCP4 suite and current developments. ActaCrystallogr Sect D: Biol Crystallogr 67:235–242.

12. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. ActaCrystallogr Sect D: Biol Crystallogr 60:2126–2132.

13. Vagin A, Teplyakov A (1997) MOLREP: An automated program for molecular replace-ment. J Appl Crystallogr 30:1022–1025.

14. Bond CS (2003) TopDraw: A sketchpad for protein structure topology cartoons. Bioin-formatics 19:311–312.

15. Rao ST, Rossmann MG (1973) Comparison of super-secondary structures in proteins.J Mol Biol 76:241–256.

16. Rossmann MG, Bernal R, Pletnev SV (2001) Combining electron microscopic with x-raycrystallographic structures. J Struct Biol 136:190–200.

17. Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: Semiautomated software for high-reso-lution single-particle reconstructions. J Struct Biol 128:82–97.

Fig. S1. The NDV nucleocapsid. (A) A repeating pattern along the length of NDV nucleocapsids was seen in the tomographic data. The image was generatedby 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 lengthof 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 of18 subunits/turn.



Fig. S2. Nonreducing, silver-stained SDS-PAGE of purified virus. The center and right lanes were loadedwith 1 and 2.5 μL of purified NDV, respectively. The leftlane was loaded with amolecular weight marker. The viral proteins and marker molecular weights are identified in the figure. Although the majority of virionsdo 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.



Fig. S3. The relationship between the matrix layer and the nucleocapsid filaments. (A and B) Tomographic sections showing portions of the nucleocapsid oftwo virions (Top). The yellow arrows indicate portions of the nucleocapsid that are roughly in register with the adjoiningmatrix array (Middle). The relationshipbetween the nucleocapsids and matrix array are shown schematically (Bottom). The yellow rectangles represent portions of the nucleocapsid and the bluesquares indicate the approximate orientation of the matrix array, the diagonal of which forms a roughly 45° angle with the long axis of elongated virions. Highdensity 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) Theaveraged 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 averagingprocedure were used to place the matrix density. The 7 nm repeating units of the matrix array and nucleocapsid structure are oriented in roughly the samedirection. 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 depicted in B. The7 nm repeating units of the matrix array and nucleocapsid structure are oriented in roughly the same direction. (F) The structures depicted in E, rotatedapproximately 90° about the horizontal axis. The scale bar represents 25 nm.



Fig. S4. Gel filtration indicates the matrix protein is dimeric. (Left) The matrix protein was purified using a Superdex 200 16/60 GL (GE Healthcare) size-exclusion column. The arrows indicate three different elution peaks for NDV matrix protein. The dashed vertical lines indicate the elution peaks of fourmolecular weight standards. A comparison between the matrix protein elution positions and the molecular weight standards indicates that the matrix proteinis mainly dimeric or forming higher-order oligomers in solution, though somemonomeric protein is present. (Inset, Right) SDS-PAGE of the protein eluted fromthe monomeric (P3), dimeric (P2), and higher-order oligomer (P1) peaks. The left lane was loaded with a molecular weight marker. Marker molecular weightsare indicated in the figure.



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 10members of the Paramyxovirinaesubfamily. 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 themonomer to the dimer interface are less conserved among paramyxoviruses.



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



Table S1. Crystallographic data collection and refinement statistics*

Native 1 Native 2 Hg derivative

Data collectionX-ray source 23-ID-D 23-ID-D 23-ID-BWavelength (Å) 1.03 1.03 1.03Cell dimensions a ¼ 163, b ¼ 47, c ¼ 117;

β ¼ 131, α ¼ γ ¼ 90a ¼ 247, b ¼ 45, c ¼ 58;

β ¼ 103, α ¼ γ ¼ 90a ¼ 46.8, b ¼ 111.8

c ¼ 141.8;α ¼ β ¼ γ ¼ 90Space group C2 C2 P22121No. monomers per asymmetric unit 2 2 2Oscillation angle 1 1 1No. frames 257 140 360Resolution range 30–2.2 30–2.2 30–2.2Rmerge

† 0.088(0.612) 0.078(0.602) 0.116(0.364)Completeness 99.6(100) 93.9(94.6) 100(100)Redundancy 5.1 2.8 14.6RefinementResolution (Å) 30–2.2 30–2.2 30–2.2Rworking

‡ (%) 24.5 23.5 22.6Rfree

§ (%) 28.8 28.6 26.3Average B factor (Å2) 55.2 54.4 28.2Rmsd bonds (Å) 0.017 0.013 0.020Rmsd angles (°) 2.189 1.787 1.979Ramachandran disallowed 0 0 0

*Values in parentheses throughout the table correspond to the last shell.†Rmerge ¼ ΣjI − hIij∕ΣI, where I is the measured intensity for reflections with indices hkl.‡Rworking ¼ ΣjjFobsj − jFcalcjj∕ΣjFobsj.§Rfree has the same formula as Rworking, except that the calculation was made with the structure factors from the test set.

Table S2. Fitting of the crystallographic matrixprotein dimer into the averaged matrix proteindensity from cryoelectron tomography using theEMfit program

Sumf* Clash† -Den‡

41.7 0.4% 8.7%

*Sumf is the mean density height averaged over allatoms where the maximum density in the electrondensity map is set to 100.

†Clash represents the percentage of atoms in the modelthat approach closer than 5 Å to neighboring matrixdimers.

‡The percentage of atoms in density less than the meandensity value is given by –Den.

Table S3. Sequence and structural comparisons between the 360 Cα atoms of the NDVmatrix protein and otherMononegavirales*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 17.9 3.0 242 254 2VQP 7.5%BDV M 5.4 2.8 109 140† 3F1J 7.8%EBV M 5.2 6.8 150 260‡ 1ES6 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.


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