Tables S1 & S2

Fig. S1. Complex purification and size-exclusion chromatography. A) SDS-PAGE analysis of EF-Tu•CdiA-CT•CdiINC101 purified by Ni2+-affinity chromatography. B) The mass of the ternary complex was estimated by size-exclusion chromatography using a Hiload 26/60 Superdex 200 column equilibrated with 20 mM sodium phosphate (pH 7.4), 150 mM NaCl at 1 mL/min. The elution profiles of the EF-Tu•CdiA-CT•CdiINC101 complex (red trace) and molecular weight standards (black trace) are shown. Mass standards are thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa), and vitamin B12 (1.35 kDa).

Fig. S2. 2DFo-mFc electron density maps. Nuclease domain in the EF-Tu2,3•CdiACT•CdiINC101 (panel A) and EF-Tutr•CdiA-CT•CdiINC101 (panel B) complexes. C) GDP bound to EF-Tu (chain A) in the EF-Tutr•CdiA-CT•CdiINC101 model. All density maps are contoured at 1σ level.

Fig. S3. Structural homologs of the CdiA-CTNC101 nuclease domain. Superimposition of CdiA-CTNC101 with A) BrnT (PDB:3U97), B) C-terminal domain of colicin D (PDB:1TFO) and C) C-terminal domain of colicin E5 (PDB:3VJ7). Functionally important residues are shown in stick representation.

Fig. S4. CdiA-CTNC101 in vivo activity screen. CdiA-CT toxins from E. coli strains NC101, 3006 and 96.154 were activated inside E. coli cells for 2 h. Cells were harvested and RNA extracted for Northern blot hybridization using oligonucleotide probes to the indicated tRNAs. Anticodon sequences are provided in parentheses to indicate specific isoacceptors. tRNAs in red font are efficiently cleaved by CdiA-CTNC101, and those in blue are subject to partial cleavage.

Fig. S5. CdiA-CTNC101 nuclease domain homologs. Homologous domains were aligned using Clustal omega and rendered using the ESPript 3.0 server 54. Sequences are from the following proteins (NCBI reference identifiers in parentheses): E. coli NC101 (WP_000554167.1), E. coli UMEA 3341-1 (WP_021571728.1), Yersinia mollaretii 107/02 (CQH18187.1), Serratia marcescens SmUNAM836 (ALE97359.1), Serratia grimesii A2 (KFB87175.1), Klebsiella pneumoniae MGH-68 (WP_064735329.1), Erwinia sp. Leaf53 (WP_056237199.1), Acetobacter tropicalis LMG 1663 (WP_061488370.1), Haemophilus influenzae 167_HINF (WP_049380275.1), Pseudomonas sp. NFACC42-2 (SFS24821.1), Pseudomonas umsongensis 20MFCvi1.1 (WP_018929888.1), and Caulobacter sp. Root655 (WP_056722815.1).

Fig. S6. Immunoblot analysis of chimeric CdiA-CTNC101 effector proteins. E. coli EPI100 cells harboring CdiAEC93-CTNC101 expression plasmids pCH11434 (wild-type), pCH12749 (H248A), pCH13355 (E236A), pCH13354 (R200A) or pCH12796 (Y192R) were treated with proteinase K where indicated, then quenched with PMSF prior to lysis. Whole cell lysates were analyzed by immunoblot using polyclonal antibodies raised against the N-terminal two-partner secretion transport domain of CdiAEC93.

Fig. S7. Protein-protein interactions. A) Purified His6-CdiA-CTNC101, EF-Tu and EF-Ts were mixed, then subjected to Ni2+-affinity chromatography under non-denaturing conditions and fractions analyzed by SDS-PAGE. The input lane represents the initial mixture prior to chromatography, the free lane shows proteins that failed to bind the column, and the bound lane shows proteins that eluted from the column with imidazole. B) Purified CdiINC101-His6, CdiACTNC101, EF-Tu and EF-Ts were mixed and analyzed as described in panel A. C) The indicated CdiA-CTNC101 variants were mixed with purified EF-Tu and CdiINC101-His6 and analyzed as described in panel A.

Table S1. Plasmids Plasmids

Descriptiona

Reference

pTrc99a

IPTG-inducible expression plasmid, AmpR

GE Healthcare

pTrc99KX

Derivative of pTrc99a that contains a 5´-KpnI restriction site and 3´-SpeI and XhoI sites, AmpR

(1)

pMCSG58

T7 promoter containing protein over-production vector, AmpR

(2)

pMCSG58CPX200205

pMCSG58::cdiA-CT/cdiINC101, AmpR

pCH405∆

pACYC184 derived cloning vector, TetR

(3)

pCH450

pACYC184 derivative with E. coli araBAD promoter for arabinose-inducible expression, TetR

(4)

pCH1043

pACYC184 derivative that expresses tRNACCUArg from native promoter, TetR

(5)

pCH2820

pCH405∆::argU, expresses tRNAUCUArg from native promoter, TetR

(5)

pCH4388

pCH405∆::tyrT, expresses tRNAQUATyr from native promoter, TetR

This study

pCH6279

pUC57::cdiA-CT/cdiINC101, AmpR

Genscript

pCH7171

pCH450::cdiA-CT/cdiIEC536(DAS), TetR

(6)

pCH10163

Cosmid pCdiA-CT/pheS* that carries a kan-pheS* cassette in place of the E. coli EC93 cdiA-CT/cdiI coding sequence. Used for allelic exchange and counter-selection. CmR KanR

(7)

pCH11434

pTNC::cdiBAEC93-CTNC101/cdiINC101, CmR

(8)

pCH12042

pTrc99KX::cdiINC101, AmpR

(8)

pCH12598

pCH450::cdiA-CT/cdiINC101-DAS, TetR

This study

pCH12641

pSH21::trxA-TEV-cdiA-CT/cdiINC101, AmpR

This study

pCH12745

pET21::cdiINC101, AmpR

This study

pCH12749

pTNC::cdiBAEC93-CT(H248A)NC101/cdiINC101, CmR

This study

pCH12796

pTNC::cdiBAEC93-CT(Y192R)NC101/cdiINC101, CmR

This study

pCH12881

pSH21::cdiA-CT/cdiINC101, AmpR

This study

pCH12935

pSH21::trxA-TEV-cdiA-CT(Y192R)/cdiINC101, AmpR

This study

pCH12936

pSH21::trxA-TEV-cdiA-CT(H248A)/cdiINC101, AmpR

This study

pCH12602

pSH21::trxA-TEV-tsf, AmpR

(9)

pCH12603

pSH21::trxA-TEV-tufA, AmpR

(9)

pCH13076

pET21K::cca-his6, AmpR

This study

pCH13321

pCH450::cdiA-CT(K186A)/cdiINC101-DAS, TetR

This study

This study

a

pCH13322

pCH450::cdiA-CT(K189A)/cdiINC101-DAS, TetR

This study

pCH13323

pCH450::cdiA-CT(K194A)/cdiINC101-DAS, TetR

This study

pCH13324

pCH450::cdiA-CT(Q198A)/cdiINC101-DAS, TetR

This study

pCH13325

pCH450::cdiA-CT(R200A)/cdiINC101-DAS, TetR

This study

pCH13326

pCH450::cdiA-CT(R222A)/cdiINC101-DAS, TetR

This study

pCH13327

pCH450::cdiA-CT(R229A)/cdiINC101-DAS, TetR

This study

pCH13328

pCH450::cdiA-CT(E231A)/cdiINC101-DAS, TetR

This study

pCH13329

pCH450::cdiA-CT(K242A)/cdiINC101-DAS, TetR

This study

pCH13330

pCH450::cdiA-CT(H248A)/cdiINC101-DAS, TetR

This study

pCH13331

pCH450::cdiA-CT(Q250A)/cdiINC101-DAS, TetR

This study

pCH13332

pCH450::cdiA-CT(K253A)/cdiINC101-DAS, TetR

This study

pCH13353

pTNC::cdiBAEC93-CT(Q198A)NC101/cdiINC101, CmR

This study

pCH13354

pTNC::cdiBAEC93-CT(R200A)NC101/cdiINC101, CmR

This study

pCH13355

pTNC::cdiBAEC93-CT(E236A)NC101/cdiINC101, CmR

This study

pCH13356

pTNC::cdiBAEC93-CT(Q250A)NC101/cdiINC101, CmR

This study

pCH13438

pCH405∆::argW(A72G), TetR

This study

pCH13439

pCH405∆::argU(G74A), TetR

This study

pCH13440

pCH405∆::tyrT(A82G), TetR

This study

pCH13479

pCH450::cdiA-CT(E236A)/cdiINC101-DAS, TetR

This study

pCH13481

pCH450::cdiA-CT(Y192R)/cdiINC101-DAS, TetR

This study

Abbreviations: AmpR, ampicillin-resistant; CmR, chloramphenicol-resistant; KanR, kanamycin-resistant;

TetR, tetracycline-resistant

References 1. 2. 3.

Koskiniemi, S., Garza-Sanchez, F., Sandegren, L., Webb, J.S., Braaten, B.A., Poole, S.J., Andersson, D.I., Hayes, C.S. and Low, D.A. (2014) Selection of orphan Rhs toxin expression in evolved Salmonella enterica serovar Typhimurium. PLoS Genet, 10, e1004255. Eschenfeldt, W.H., Makowska-Grzyska, M., Stols, L., Donnelly, M.I., Jedrzejczak, R. and Joachimiak, A. (2013) New LIC vectors for production of proteins from genes containing rare codons. J Struct Funct Genomics, 14, 135-144. Hayes, C.S., Bose, B. and Sauer, R.T. (2002) Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli. Proc Natl Acad Sci U S A, 99, 3440-3445.

4. 5. 6. 7. 8. 9.

Hayes, C.S. and Sauer, R.T. (2003) Cleavage of the A site mRNA codon during ribosome pausing provides a mechanism for translational quality control. Mol. Cell, 12, 903-911. Garza-Sánchez, F., Gin, J.G. and Hayes, C.S. (2008) Amino acid starvation and colicin D treatment induce A-site mRNA cleavage in Escherichia coli. J Mol Biol, 378, 505-519. Johnson, P.M., Beck, C.M., Morse, R.P., Garza-Sanchez, F., Low, D.A., Hayes, C.S. and Goulding, C.W. (2016) Unraveling the essential role of CysK in CDI toxin activation. Proc Natl Acad Sci U S A, 113, 9792-9797. Morse, R.P., Nikolakakis, K.C., Willett, J.L., Gerrick, E., Low, D.A., Hayes, C.S. and Goulding, C.W. (2012) Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems. Proc Natl Acad Sci U S A, 109, 21480-21485. Willett, J.L., Gucinski, G.C., Fatherree, J.P., Low, D.A. and Hayes, C.S. (2015) Contactdependent growth inhibition toxins exploit multiple independent cell-entry pathways. Proc Natl Acad Sci U S A, 112, 11341-11346. Jones, A.M., Garza-Sanchez, F., So, J., Hayes, C.S. and Low, D.A. (2017) Activation of contactdependent antibacterial tRNase toxins by translation elongation factors. Proc Natl Acad Sci U S A, 114, E1951-E1957.

Table S2. Oligonucleotides Identifier

Descriptive name

Sequence

PSI-1

NC101-CT-for

5´ - GTC TCT CCC ATG GTT GAG AAT AAC TAC CTG AGC GTG TCT

PSI-2

NC101-cdiI-rev

5´ - TGG TGG TGC CCA GCT TCA GGC CAT GCC AAT CCA TCA CT

CH367

Gly-GCC-probe

5´ - CTT GGC AAG GTC GTG CT

CH368

Pro-GGG-probe

5´ - CAC CCC ATG ACG GTG CG

CH374

Ala-UGC-probe

5´ - TCC TGC GTG CAA AGC AG

CH379

Arg-CCU-probe

5´ - CCT GCA ATT AGC CCT TAG G

CH380

Gly-UCC-probe

5´ - CCC GCA TCA TCA GCT TGG AAG GC

CH381

Leu-CAA-probe

5´ - CCC GCA CAG CGC GAA CGC CG

CH382

Phe-GAA-probe

5´ - TGC TCT ACC GAC TGA GCT A

CH383

Pro-CGG-probe

5´ - CTT CGT CCC GAA CGA AGT G

CH385

Thr-CGU-probe

5´ - CCT ACG ACC TTC GCA TT

CH436

Arg-CCG-probe

5´ - CCT GAG ACC TCT GCC TCC GGA

CH451

Arg-UCU-probe

5´ - CCT GCG GCC CAC GAC TTA G

CH452

Arg-ICG-probe

5´ - CCT CCG ACC GCT CGG TTC G

CH487

Cys-GCA-probe

5´ - GGA CTA GAC GGA TTT GCA A

CH577

Ile-GAU-probe

5´ - ACC GAC CTC ACC CTT ATC AG

CH618

Trp-CCA-probe

5´ - CCC AAC ACC CGG TTT TGG

CH791

Gln-CUG-probe

5´ - TCG GAA TGC CGG AAT CAG A

CH798

Tyr1-GUA-probe

5´ - CTT CGA AGT CTG TGA CGG CAG

CH799

Tyr2-GUA-probe

5´ - CTT CGA AGT CGA TGA CGG CAG

CH800

Asp-QUC-probe

5´ - TCG AAC CCG CGA CCC CCT GCG

CH801

Asn-QUU-probe

5´ - CTC GAA CCA GTG ACA TAC GG

CH837

Ser-GCU-probe

5´ - CCC CGG ATG CAG CTT TTG ACC

CH1046

Leu-GAG-probe

5´ - CCC GTA AGC CCT ATT GGG CA

CH1047

Ser-UGA-probe

5´ - AAC CCT TTC GGG TCG CCG GTT TTC

CH1248

Val-UAG-probe

5´ - CGC CGA CCC CCT CCT TGT AAG

CH1249

Lys-UUU-probe

5´ - CCT GCG ACC AAT TGA TTA AA

CH1417

Glu-UUC-probe

5´ - CCC CTG TTA CCG CCG TG

CH1737

3´-T7-glnX

5´ - TGG CTG GGG TAC GAG GAT TCG AAC CTC GGA ATG CCG GAA TCA GAA TCC GGT GCC TT

CH2032

Gly-CCC-probe

5´ - CCC TCG TAT AGA GCT TGG GAA

CH2034

Leu-CAG-probe

5´ - CCC CCA CGT CCG TAA GGA CA

CH2035

Leu-UAG-probe

5´ - CAC CTT GCG GCG CCA GAA

CH2036

Leu-UAA-probe

5´ - CCC GCA CAG CGC GAA CGC CG

CH2037

fMet-CAU-probe

5´ - CGG GTT ATG AGC CCG ACG A

CH2038

Met-CAU-probe

5´ - CCT GTG ACC CCA TCA TTA TGA

CH2040

Ser-CGA-probe

5´ - GTA GAG TTG CCC CTA CTC CGG

CH2042

Thr-GGU-probe

5´ - CTG GGG ACC CCA CCC CT

CH3176

NC101-CT(OE)-for

5´ - CAG GTA GGA ACT CGG TTG AGA ATA ATT ACC TGA GCG TGT CTG AAA AGA CAG

CH3177

NC101-CT(OE)rev

5´ - GGT CTG GTG TCT AAC CTT TGG GTT ATT CAG GCC ATG CCA ATC CAT C

CH3238

NC101-cdiI-Kpnfor

5´ - CTG GTA CCA TGG ATA TTT GGC CTG

CH3239

NC101-cdiI-Xhorev

5´ - GAC TCG AGT TAT TCA GGC CAT GCC AAT C

CH3269

PSI-univ-Nco-for

5´ - TTT AAG AAG GAG TCT CTC CCA TGG

CH3422

NC101-cdiI-Sperev

5´ - GAA CTA GTT TCA GGC CAT GCC AAT C

CH3880

NC101-CT-Kpn-for

5´ - CTG GTA CCG TTG AGA ATA ACT ACC TGA GC

CH3884

NC101-H248A-for

5´ - GGG ACA AGT TGG TGC TAT TCA ACC AGT CAA G

CH3885

NC101-H248AXho-rev

5´ - TTC CTC GAG CTC TTTT AAC CTT GAC TGG TTG AAT AGC ACC AAC TTG TCC C

CH3891

NC101-CT-Spe-for

5´ - TTT ACT AGT GTT GAG AAT AAC TAC C

CH3905

NC101-Y192R-for

5´ - CAG CAA AAG TTA TCG TGA GAA ATT ATG GG

CH3916

gltT S1 probe

5´ - TGG CGT CCC CTA GGG GAT TCG AAC CCC TGT TAC CGC CGT G

CH3917

gltT G67 marker

5´ - CTA GGG GAT TCG AAC CCC TGT TAC CGC CGT G

CH3918

gltT G70 marker

5´ - CCC CTA GGG GAT TCG AAC CCC TGT TAC CGC CGT G

CH3919

gltT G73 marker

5´ - CGT CCC CTA GGG GAT TCG AAC CCC TGT TAC CGC CGT G

CH4013

5´-T7-tRNA-Asp

5´- AAT TCC TGC AGT AAT ACG ACT CAC TAT AGG AGC GGT AGT TCA GTC GGT TAG AAT ACC TG

CH4014

3´-tRNA-Asp

5´- TGG CGG AAC GGA CGG GAC TCG AAC CCG CGA CCC CCT GCG TGA CAG GCA GGT ATT CTA AC

CH4062

cca-Kpn-for

5´ - GAT GGT ACC ATG AAG ATT TAT CTG GTC

CH4063

cca-Xho-rev

5´ - CAA CTC GAG TTC AGG CTT TGG GCA ACG

CH4100

EC93-cdiA-8220for

5´ - GAA GCG ATG AAA GCA GCC AGG

CH4101

EC93-cdiA-8718rev

5´ - CTC AAC CGA GTT CCT ACC TGC CTG

CH4102

EC93-cdiI-296-for

5´ - AAC CCA AAG GTT AGA CAC CAG ACC

CH4103

EC93-cdiI-+486rev

5´ - AAG TAG GCA TTC TCG ACC CTG

CH4138

NC101-E236A-for

5´ - GGT GCA GGT CTT GCA ATG ATC TAT AAC C

CH4178

argU-G74A-for

5´ - CCT GCA GGG CGC ACC ATT ACA ATT C

CH4179

argW-A72G-for

5´ - CGA TTC CTG CAG GGG ACG CCA TTT ATC AGT TCG

CH4180

tyrT-A82G-for

5´ - GAA TCC TTC CCC CAC CGC CAT AAT TCA CCA CAG

CH4181

argU-G74A-rev

5´ - GAA TTG TAA TGG TGC GCC CTG CAG G

CH4182

argW-A72G-rev

5´ - CGA ACT GAT AAA TGG CGT CCC CTG CAG GAA TCG

CH4183

tyrT-A82G-rev

5´ - CTG TGG TGA ATT ATG GCG GTG GGG GAA GGA TTC

CH4437

3´-tRNA-Asp∆CCA

5´ - CGG AAC GGA CGG GAC TC GAA CCC GCG ACC CCC TGC GTG ACA GGC AGG TAT TCT AAC

NC101-K186A-for

5´ - CTG TAG ATG GAA TGG CAT TCA GCA AAA GT

NC101-K186A-rev

5´ - ACT TTT GCT GAA TGC CAT TCC ATC TAC AG

NC101-K189A-for

5´ - GGA ATG AAA TTC AGC GCA AGT TAT TAT GAG A

NC101-K189A-rev

5´ - TCT CAT AAT AAC TTG CGC TGA ATT TCA TTC C

NC101-K194A-for

5´ - CAA AAG TTA TTA TGA GGC ATT ATG GGA GCA AGG C

NC101-K194A-rev

5´ - GCC TTG CTC CCA TAA TGC CTC ATA ATA ACT TTT G

NC101-Q198A-for

5´ - GAA ATT ATG GGA GGC AGG CAG ACC TGC

NC101-Q198A-rev

5´ - GCA GGT CTG CCT GCC TCC CAT AAT TTC

NC101-R200A-for

5´ -GAA ATT ATG GGA GCA AGG CGC ACC TGC GCC ATT CGT GCA AG

NC101-R200A-rev

5´ - CTT GCA CGA ATG GCG CAG GTG CGC CTT GCT CCC ATA ATT TC

NC101-R222A-for

5´ - AAA TTG AGC CGG ATC CTG CAG GCG CAC CTG GCT AC

NC101-R222A-rev

5´ - GTA GCC AGG TGC GCC TGC AGG ATC CGG CTC AAT TT

NC101-R229A-for

5´ - CCT GGC TAC TTA GCA TAT GAA GGT GC

NC101-R229A-rev

5´ - GCA CCT TCA TAT GCT AAG TAG CCA GG

NC101-E231A-for

5´ - CTA CTT AAG ATA TGC TGG TGC AGG TCT TG

NC101-E231A-rev

5´ - CAA GAC CTG CAC CAG CAT ATC TTA AGT AG

NC101-K242A-for

5´ - GAT CTA TAA CCC GGC AAC GGG ACA AGT TG

NC101-K242A-rev

5´ - CAA CTT GTC CCG TTG CCG GGT TAT AGA TC

NC101-Q250A-for

5´ - GTT GGT CAT ATT GCA CCA GTC AAG G

NC101-Q250A-rev

5´ - CCT TGA CTG GTG CAA TAT GAC CAA C

NC101-K253A-for

5´ - GGT CAT ATT CAA CCA GTC GCG GTT AAA TAA AAA GAG G

NC101-K253A-rev

5´ - CCT CTT TTT ATT TAA CCG CGA CTG GTT GAA TAT GAC C

Table S3. Pair-wise comparisons of chains from the two EF-Tu•CdiA-CT•CdiINC101 structural models. Root-mean-square deviations (rmsd) are given in Å. The number of superimposed Ca atoms is given in parentheses.

EF-Tu2,3•CT•CdiI A (CdiA-CT)

B (CdiI)

C (EF-Tu2,3)

ABC

A (CdiA-CT)

0.116 (87)

–

–

–

B (CdiI)

–

0.207 (106)

–

–

CD (EF-Tu )

–

–

0.455 (186)

–

E (CdiA-CT)

0.084 (86)

–

–

–

F (CdiI)

–

0.194 (106)

–

–

GH (EF-Tu )

–

–

0.366 (186)

–

ABCD

–

–

–

0.458 (379)

EFGH

–

–

–

0.350 (378)

EF-Tutr•CT•CdiI

tr

tr

Table S4. Structural homologs of CdiA-CTNC101 and CdiINC101 Protein

Homolog (organism)

PDB IDa

Zscore

rmsdb (Å)

lali/nresc

% identity

CdiA-CTNC101

BrnT (B. abortus)

3U97 (A)

7.1

2.4

67/77

13

colicin D (E. coli)

1V74 (A)

5.4

3.8

74/107

11

colicin D (E. coli)

1TFO (A)

5.3

3.2

72/103

11

colicin E5 (E. coli)

2DJH (A)

4.7

2.8

62/96

16

colicin E5 (E. coli)

3VJ7 (A)

4.7

2.7

61/94

15

colicin E5 (E. coli)

3AO9 (B)

4.6

2.8

62/86

16

colicin D (E. coli)

1TFK (A)

4.5

3.2

64/94

13

MqsR (E. coli)

3HI2 (D)

4.5

3.2

64/91

8

BAG1 (H. sapiens)

5AQQ (B)

6.7

3.3

82/106

11

NEQ131 (N. equitans)

5JRE (D)

6.4

2.9

84/185

8

AT1BAG (A. thaliana)

4HWI (B)

6.1

2.0

84/177

5

GGA1 (H. sapiens)

1OXZ (A)

5.9

3.6

85/132

11

CdiINC101

a

Structurally homologous chain is given in parentheses.

b

root-mean-square deviation.

c

length of alignment (lali)/total number of residues (nres)

Table S5. Direct H-bond and ion-pair interactions. Interactions are grouped for each protein-protein interface with EF-Tu/CdiA-CTNC101 highlighted in orange, CdiA-CT/CdiINC101 in green, and EFTu/CdiINC101 in cyan. B´ indicates the (y, x, -z) symmetry related copy of CdiINC101. H-bond donor

H-bond acceptor

Distance (Å)

Chain

Residue/atom

Chain

Residue/atom

A (CdiA-CT)

Lys171 [N]

C (EF-Tu)

Leu266 [O]

2.90

Asp267 [OD2] Lys264 [O] Glu260 [OE2] Glu260 [OE1] Glu260 [OE1] Asp267 [OD1] Asp267 [OD2] Asp267 [OD1] Glu209 [OE1] Glu209 [OE1] Tyr192 [OH] Lys171 [O]

3.10 2.83 2.52 2.85 3.42 3.52 2.79 2.81 2.74 3.27 2.93 2.77

Glu236 [OE1] Glu236 [OE2] Asp220 [OD1] Asp220 [OD2] Asp220 [OD2] Ser59 [OG] Asp84 [OD1] Asp84 [OD2] Asp84 [OD2] Tyr81 [O] Glu288 [OE1] Thr336 [OG]

2.69 2.94 2.77 3.46 2.81 3.02 3.20 3.18 2.52 3.18 2.68 2.96

Glu288 [OE1] Thr336 [OG] Asp337 [O] Asp217 [OD2] Glu69 [OE2] Glu69 [OE1] Glu6 [OE1]

3.17 3.03 3.03 3.45 2.82 2.87 2.69

C (EF-Tu)

B (CdiI)

A (CdiA-CT)

B´ (CdiI)

B (CdiI) C (EF-Tu)

Lys171 [NZ] Ile173 [N] Tyr192 [OH] Phe204 [N] Val205 [N] Arg208 [NH1] Arg208 [NH1] Arg208 [NH2] Arg224 [NE] Arg224 [NH2] Arg263 [N] Leu266 [N] Tyr81 [OH] Tyr81 [OH] Arg85 [NH1] Arg85 [NH1] Arg85 [NH2] Lys194 [NZ] Arg200 [NH1] Arg200 [NH2] His248 [NE2] Gln250 [NE2] Arg23 [NE] Arg23 [NH1] Arg23 [NH2] Arg23 [NH2] Arg23 [NH1] Lys67 [NZ] Arg284 [NE] Arg284 [NH2] Arg328 [NH1]

A (CdiA-CT)

A (CdiA-CT)

B (CdiI)

C (EF-Tu)

B (CdiI)