SUPPLEMENTARY INFORMATION

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1 In the format provided by the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 2 ARTICLE NUMBER: Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single particle analysis Katherine S.H. Beckham 1#, Luciano Ciccarelli 2,3,4#, Catalin M. Bunduc 5#, Haydyn D.T. Mertens 1, Roy Ummels 6, Wolfgang Lugmayr 2,3,4, Julia Mayr 2,3,4, Mandy Rettel 7, Mikhail M. Savitski 7, Dmitri I. Svergun 1, Wilbert Bitter 5,6, Matthias Wilmanns 1,4,8, Thomas C. Marlovits 2,3,4,8,9*, Annabel H.A. Parret 1*, Edith N.G. Houben 5* # equal contribution * Correspondence: Thomas.Marlovits@imba.oeaw.ac.at, parret@embl-hamburg.de, e.n.g.houben@vu.nl 1 European Molecular Biology Laboratory, Hamburg Unit, Notkestraße 85, 22607, Hamburg, Germany 2 Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria 3 Institute of Molecular Biotechnology, Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria 4 University Medical Centre Hamburg-Eppendorf, Martinistraße 52, Hamburg, Germany 5 Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands 6 VU University Medical Center, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands 7 European Molecular Biology Laboratory, Meyerhofstraße 1, Heidelberg, Germany 8 Centre for Structural Systems Biology, Notkestraße 85, Hamburg, Germany 9 Deutsches Elektronen-Synchrotron, Notkestraße 85, Hamburg, Germany This file contains Supplementary Figure 1-9 and Supplementary Tables 1-3. NATURE MICROBIOLOGY DOI: /nmicrobiol Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Supplementary Figure 1. Conserved gene architecture and expression of esx-5 loci. a, Gene organization of the esx-5 locus in M. tuberculosis H37Rv. The homologous esx-5 locus from M.

2 Supplementary Figure 1. Conserved gene architecture and expression of esx-5 loci. a, Gene organization of the esx-5 locus in M. tuberculosis H37Rv. The homologous esx-5 locus from M. xenopi was expressed in M. smegmatis from the pmv-esx-5 construct. Genes have been coloured according the scheme shown in Fig. 3g. The Strep-tag II on eccc5 is shown as a black box. To test the effect of secreted effectors on complex formation, all genes encoding for substrates (two pe, two ppe and two esx genes) were deleted to generate pmv-esx-5 substr as indicated by the dotted lines. b, Immunoblot of detergent-solubilized cell envelope fractions of M. smegmatis expressing pmv-esx-5 and pmv-esx-5 substr analysed by blue native PAGE. Intact ESX-5 complex is indicated by a black arrow. Representative immunoblot using anti-eccb 5 antibodies from at least three independent experiments.

a 60 670 158 44 17 1.4 b DDM A8-35 c A8-35 DDM standards Normalized mau (280 nm) 40 20 1236-1048 - 720-480 - 146 - d 0 5 10 15 20 25 Volume (ml) 66 - Supplementary Figure 2.

$The arrow indicates the elution fraction used for blue native PAGE and EM analysis.$

3 a b DDM A8-35 c A8-35 DDM standards Normalized mau (280 nm) d Volume (ml) 66 - Supplementary Figure 2. Improved stability of the ESX-5 complex in amphipols. a, Analytical size exclusion chromatography analysis of the ESX-5 complex in DDM (grey line) versus amphipol A8-35 (black line). The arrow indicates the elution fraction used for blue native PAGE and EM analysis. The molecular weight of a standard set of protein markers is indicated on top of the corresponding peak (dotted line; in kilodaltons). b. Coomassiestained blue native PAGE gel of ESX-5 complex solubilized in DDM or A8-35. Representative gel from at least three independent experiments. c, and d, Representative negative stain EM showing ESX-5 particles solubilized in DDM and A8-35, respectively. Scale bar is 50 nm.

a b c 1.0 d 50º 0º 0.8 0.6 CC FSC 0.4 0.2 FSC = 0.143 13.1 Å Degrees 0.0 0.0 0.05 0.

a, Gallery of representative 2D class averages. b, Rotational cross-correlation (CC) from a representative 2D class average. c, Fourier Shell Correlation curve of the ESX-5 complex reconstruction.

e, Angular distribution applying C6 symmetry is shown by the graphical display of e2eulerxplor.py.

4 a b c 1.0 d 50º 0º CC FSC FSC = Å Degrees Resolution (1/Å) e f g 90º 150º 100º 50º 180º 0º Untilted Tilted Untilted Tilted Untilted Tilted 270º Supplementary Figure 3. Single particle analysis of ESX-5 complexes and map validation. a, Gallery of representative 2D class averages. b, Rotational cross-correlation (CC) from a representative 2D class average. c, Fourier Shell Correlation curve of the ESX-5 complex reconstruction. The resolution at was 13.1Å. d, Upper panel shows a representative tilt-pair of ESX-5 particles at 0 and 50 tilting. Below are the corresponding views of the 3D map. e, Angular distribution applying C6 symmetry is shown by the graphical display of e2eulerxplor.py. Projections in top, intermediate and side views are shown on the left row adjacent to the corresponding 2D class average. Particle orientation is evenly distributed in the 3D space extending from the equator to the pole, enabling a robust negative stain 3D reconstruction with no missing information. f, Representative tilt-pairs collected at 0 and 50 and subjected to tilt-pair analysis. g, Tilt-pair parameter plot (TPPP) 1 of tilt-pairs shows a clear clustering around the 50 tilt angle corresponding to the angle that was used to record the second micrograph of the tilt pair.

5 a Absorbance at 280 nm (mau) EccC 5 99 Theoretical MW (Da) Experimental MW (Da) Monomer 142, ,971 Dimer 284, , Molecular weight (Da) Retention volume (ml) b c P(r) P(r) log log I(s) I(S) DUF A1 A2 A3 Χ D max (nm) r (nm) s (nm -1 ) Supplementary Figure 4. Analysis of truncated EccC5 (EccC5 99 ) by SEC-MALS and SAXS. a, Molecular weights were calculated from analytical size-exclusion chromatography analysis coupled with static light scattering and plotted across the peaks (blue). The derived molar weights of 147 and 310 match the expected molecular weights of a monomeric and dimeric protein, respectively. b, Pair-wise distance distribution (P(r)) of the scattering data. c, Fits of model ensemble 3 to the raw SAXS data. Inset shows the schematic overview of modelling approaches used with the Ensemble Optimization Method (EOM). Residue boundaries of EccC 5 domains are as follows: DUF, ; A1, ; A2, ; A3, Domains boxed by dotted lines were treated as rigid bodies. The chi-squared ( 2 ) values of each of the EOM models are indicated.

a b Approach 1 Approach 2 i) Modelling EccB 5 hexamer i) Segmentation analysis Top Side Bottom Top Side Side cross-section ii) Docking EccB 5 hexamer ii) Docking EccB 5 monomer 90 Side Top Top Side

6 a b Approach 1 Approach 2 i) Modelling EccB 5 hexamer i) Segmentation analysis Top Side Bottom Top Side Side cross-section ii) Docking EccB 5 hexamer ii) Docking EccB 5 monomer 90 Side Top Top Side Side cross-section iii) Applying 6-fold symmetry Top Side Side cross-section Supplementary Figure 5. Overview outlining EccB5 docking procedures. a, Approach 1 used a hexameric model of EccB5 of M. xenopi (i) generated using the method described by Zhang et al. 2. Subsequently, the EccB5 hexamer was docked into the EM map as a rigid body (ii). Top, side and cross section views are indicated. b, In approach 2, the EM map was segmented using the program Segger revealing a feature consistent with an EccB5 hexamer (i). Next, individual EccB5 monomers were docked into the segmented volume (ii) and a sixfold symmetry was applied (iii). Both docking approaches place the N-terminus of EccB5 at the apex of the map, but differ in their positioning of the C-terminal region of EccB5 preceding the transmembrane domain.

a b c 20-250- EccC 5 strep EccB 5 EccE 5 his EccD 5 1238-1048- 720-480- 130-100- 70-55- Supplementary Figure 6. Purification of M. xenopi ESX-5 EccE-His complex.

Representative gel from at least three independent experiments. b, Coomassie-stained blue native PAGE gel of the purified complex as indicated by the black arrow.

7 a b c EccC 5 strep EccB 5 EccE 5 his EccD Supplementary Figure 6. Purification of M. xenopi ESX-5 EccE-His complex. a, Coomassiestained SDS-PAGE gel of purified ESX-5 complex following affinity purification using the Strep-tag II on EccC5 and size-exclusion chromatography. Representative gel from at least three independent experiments. b, Coomassie-stained blue native PAGE gel of the purified complex as indicated by the black arrow. Representative gel from at least three independent experiments. c, Representative negative stain EM of the ESX-5 complex. Visual inspection of the particles suggests that the overall architecture of ESX-5 EccE-His is comparable to the ESX-5 complex, indicating that the addition of a C-terminal His6-tag on EccE5 has no impact on complex formation or stability. Scale bar is 50 nm.

8 M. xenopi - DTT + DTT 75 - DTT + DTT 50 M. tuberculosis αeccb5 50 αecce5 37 Supplementary Figure 7. Disulphide bridge formation of EccE5 indicative of its periplasmic location. Immunoblot analysis of purified ESX-5 complexes under reducing (+ DTT) and nonreducing (- DTT) conditions to analyse disulphide bridge formation. This experiment was also performed on the ESX-5 system from M. tuberculosis reconstituted in M. smegmatis because of the higher number of cysteines in EccE5 from M. tuberculosis (6) versus EccE5 from M. xenopi (3). As a positive control EccB5, that is known to form a disulfide bridge 3, was also examined. Both EccB5 and EccE5 showed a difference in mobility depending on the presence of DTT. Representative immunoblots using anti-eccb5 and anti- EccE5 antibodies from three independent experiments.

the absence of EccE5. Representative gel from at least three independent experiments. b, Immunoblot of blue native PAGE gel of purified ESX-5 ΔEccE complex indicating a molecular mass of ~ 1,3 MDa.

9 Supplementary Figure 8. Purification of M. xenopi ESX-5 ΔEccE complex. a, Coomassie-stained SDS-PAGE gel of purified complex following affinity purification and size-exclusion chromatography, confirming the presence of the three components EccB5, EccC5 strep, EccD5 and the absence of EccE5. Representative gel from at least three independent experiments. b, Immunoblot of blue native PAGE gel of purified ESX-5 ΔEccE complex indicating a molecular mass of ~ 1,3 MDa. Representative immunoblot using anti-eccb5 antibodies from at least three independent experiments. c, Representative size exclusion chromatography profile of ESX-5 ΔEccE. The yield of the ESX-5 ΔEccE complex is considerably lower than that of ESX-5. The black arrow indicates the fraction shown in panel d. d, Representative electron micrograph showing ESX-5 ΔEccE particles in several orientations. The overall diameter of the particles is ~22-25nm. Scale bar is 50 nm. Representative particles of the ESX-5 ΔEccE (e) and the ESX-5 complex (f) (box size = 180 x 180 pixels). g, Gallery of class averages of 684 ESX-5 ΔEccE particles. h, Four class averages of ESX-5 and ESX-5 ΔEccE complexes rotationally aligned are shown at same magnification. i, Histogram showing the average diameter for both ESX-5 and ESX-5 ΔEccE complexes. Particles were measured along the horizontal axis, the average diameter of the ESX-5 and ESX-5 ΔEccE complexes is 28.9 ± 1.2 nm and 23.8 ± 1.3, respectively. Error bars indicate the standard deviation of 50 particles. T-test analysis shows a significant difference with a p-value

αppe18-ha 25-20 - 300 sec exposure 37-15 - αesxn 25-10

10 Full blots of results Figure 1a P S ESX-5 P S ESX αgroel αeccb5 αppe18-ha sec exposure αesxn sec exposure Figure 1b

1b ESX-5 ESX-5 Δsubstr 1236-1048 - 720-480

11 Full blots of results (continued) Supplementary Fig. 1b ESX-5 ESX-5 Δsubstr αeccb Supplementary Fig. 2b A8-35 DDM

12 Full blots of results (continued) Supplementary Fig. 6a Supplementary Fig. 6b

xenopi - + DTT - + DTT 250-250 - 150-100

13 Full blots of results (continued) Supplementary Fig. 7 M. xenopi - + DTT - + DTT αeccb5 αecce5 M. tuberculosis - + DTT - + DTT αeccb5-37 αecce5

14 Full blots of results (continued) Supplementary Fig. 8a Supplementary Fig. 8b

15 Supplementary Table 1 Quantitative mass-spectrometry analysis of ESX-5 to determine subunit stoichiometry. Average relative protein concentrations of the four subunits was determined using ibaq-based quantitative mass spectrometry and calculated across four biological replicates (1-4). These date indicate a 1:1:1:1 stoichiometry for EccB 5:EccC 5 :EccD 5:EccE 5. Proteins ibaq Relative protein concentration Average relative protein concentration S.E. Razor + unique peptides Sequence coverage (%) MW () EccB 5 3.5E E E E EccC 5 4.5E E E E EccD 5 5.2E E E E EccE 5 4.1E E E E

16 Supplementary Table 2 EM data collection and structural analysis of ESX-5. Micrographs 65 Particles 1,418 Defocus um Detector FEI BM Eagle Pixel size 2.18 Å Reconstruction method EMAN2 - e2initialmodel Symmetry 6-fold Resolution FSC gold standard 13.1 Å

17 Supplementary Table 3 SAXS data collection and derived parameters for EccC5 99. Data collection parameters Instrument EMBL P12 beam line (PETRA-III, DESY, Hamburg) Beam geometry 0.2 x 0.12 mm 2 Wavelength (Å) 1.24 s range (Å -1 ) a Exposure time (s) 1 (20 x 0.05) Concentration range (mg/ml) Temperature (K) 283 Structural parameters b I(0) (relative) (from p(r)) ± Rg (Å) (from p(r)) 65 ± 1 I(0) (relative) (from Guinier) ± Rg (Å) (from Guinier) 62 ± 1 Dmax (Å) 230 Porod volume estimate (Å 3 ) 255,000 ± 30,000 Excluded volume estimate (Å 3 ) 340,000 ± 50,000 Dry volume calculated c from sequence (Å 3 ) 172,365 Molecular mass determination I(0) (cm -1 ) BSA (72,000 Da) ± Molecular mass Mr (Da) (from I(0)) 141,000 ± 15,000 Molecular mass Mr (Da) (from Porod volume (Vp/1.7)) 159,000 ± 15,000 Molecular mass Mr (Da) (from excluded volume (Vex/2)) 173,500 ± 20,000 Calculated monomeric Mr (from sequence) 142,450 Software employed Primary data reduction Data processing Equilibrium analysis Computation of model intensities 3D graphics representations a Momentum transfer s = 4πsin(θ)/λ b Values reported for 0.4 mg ml -1 RADAVER PRIMUS/Qt EOM CRYSOL PyMOL, UCSF Chimera c Dry volume from: Abbreviations: Mr : Molecular mass Rg : Radius of gyration Dmax : Maximal particle dimension Vp : Porod volume Vex : Particle excluded volume

18 Supplementary References 1. Henderson, R. et al. Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy. J. Mol. Biol. 413, (2011). 2. Zhang, X.-L. et al. Core component EccB1 of the Mycobacterium tuberculosis type VII secretion system is a periplasmic ATPase. FASEB J. 29, (2015). 3. Wagner, J. M. et al. Structures of EccB1 and EccD1 from the core complex of the mycobacterial ESX-1 type VII secretion system. BMC Struct. Biol. 16, 5 (2016).

Supplementary figure 1. Comparison of unbound ogm-csf and ogm-csf as captured in the GIF:GM-CSF complex. Alignment of two copies of unbound ovine

Supplementary figure 1. Comparison of unbound and as captured in the GIF:GM-CSF complex. Alignment of two copies of unbound ovine GM-CSF (slate) with bound GM-CSF in the GIF:GM-CSF complex (GIF: green,