SUPPLEMENTARY INFORMATION

A structural basis for streptomycin-induced misreading of the genetic code

Hasan Demirci1, Frank Murphy IV2, Eileen Murphy1, Steven T. Gregory1, Albert E. Dahlberg1 and Ger-wald Jogl1, 3

1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912 USA 2NE-CAT/Cornell University, Argonne, IL 60439 3Corresponding author Phone: 1-401-863-6123 Fax: 1-401-863-6114 E-mail: Gerwald_Jogl@brown.edu

Supplementary Figures

Supplementary Figure S1. Superposition of equivalent 30S structures

a) Comparison of the decoding site in the Apo structure with the equivalent PDB entry 1J5E47. Only the engaged helix 45 conformation is shown for the Apo structure. b) Comparison between the MAP decoding site and the equivalent PDB entry 1IBL48.

Supplementary Figure S2. Multiple paromomycin binding sites in the bacterial 30S subunit a. Location of secondary paromomycin binding sites in the 30S subunit. Paromomycin molecules (in stick and sphere representation) observed in both data sets are colored in red. Three binding sites (colored in blue) were only observed in one data set. Panels 1 - 18. σA-weighted mFo-DFc omit difference electron density maps of paromomycin molecules contoured at 3σ. Numbering corresponds to the location indicated in (a).

Supplementary Figure S3. Distances between 16S rRNA phosphate atoms.

Pairwise distances between 16S rRNA phosphate atoms after alignment of all phosphate atoms between bases 5 and 1532 between to 30S structures. The position of the head displays higher variability in 30S crystal structures. Therefore, we focus on the shoulder region (helices 16 – 18, bases 400 -450) as an indi-cation of domain closure movements. A displacement of the entire region by about 2 Å is observed when comparing data sets MAP and Apo, or the equivalent PDB entries 1IBL48 and 1J5E47, or after domain clo-sure observed in the 70S ribosome in PDB entries 3I8G and 3I9B49. The conformational change in the helix 45 tetraloop (bases 1516 – 1521) can be seen as spike in the charts Strep-Apo and ASL Leu-Apo.

Supplementary Figure S4

Shine-Dalgarno helix formation upon interaction of a second ASL molecule (colored in green) with the 16S 3’ tail (colored in blue). σA-weighted 2mFo-DFc electron density is shown contoured at the 1σ level.

Supplementary Table S1 Data collection and refinement statistics Apo Paromomycin Streptomycin MAP (mRNA-

ASL -Par) PDB code 4DR1 4DR2 4DR3 4DR4 Data collection1 Space group P41212 P41212 P41212 P41212 Cell dimensions a, b, c (Å) 403.0, 403.0, 174.4 402.6, 402.6, 177.6 404.0, 404.0, 173.5 401.0, 401.0, 176.7 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å)2 35.0-3.6 (3.73-3.6) 45.0-3.25 (3.37-

3.25) 35.0-3.35 (3.47-

3.35) 50-3.97 (4.04-3.97)

Rmerge 0.085 (0.92) 0.115 (0.811) 0.06(0.634) 0.25 (0.99) I / σI 15.4 (1.81) 10.8 (1.81) 15.5 (1.94) 10.6 (2.55) Completeness (%) 99.2 (99.2) 99.6 (100.0) 98.3 (99.0) 100.0 (100.0) Redundancy 4.8 (4.8) 3.7 (3.8) 3.3 (3.3) 7.3 (6.7) Refinement Resolution (Å) 35-3.6 (3.64-3.60) 45-3.25 (3.29-3.25) 35-3.35 (3.39-3.35) 50-3.97 (4.01-3.97) No. reflections 163397 (5075) 226222 (7113) 200881 (6238) 123736 (3867) Rwork / Rfree 0.157/0.207

(0.280/0.324) 0.171/0.211

(0.298/0.355) 0.168/0.206

(0.290/0.345) 0.153/0.212

(0.223/0.303) No. atoms Protein 19090 19145 19090 19154 RNA 32644 32504 32507 32880 Ligand/Ion/Water 493 1578 567 1617 B-factors Protein 173.5 98.1 153.7 101.9 RNA 165.1 98.5 143.8 100.6 Ligand/Ion/Water 131.0 97.5 121.6 102.7 R.m.s deviations Bond lengths (Å) 0.012 0.007 0.006 0.013 Bond angles (°) 1.50 1.03 0.96 1.527 Ramachandran plot Favored (%) 1804 (88.0) 1874 (90.8) 1828 (89.2) 1833 (88.7) Allowed (%) 224 (10.9) 159 (7.7) 199 (9.7) 208 (10.1) Generously al-lowed (%)

20 (1.0) 32 (1.5) 22 (1.1) 24 (1.2)

Disallowed (%) 2 (0.1) 0 (0.0) 1 (0.0) 1 (0.0) 1. One crystal used for each dataset. 2. Highest resolution shell is shown in parenthesis.

Supplementary Table S1 Data collection and refinement statistics cont.

Sm-ASL -mRNA Sm-ASL -mRNA Sm-ASL -mRNA PDB code 4DR5 4DR6 4DR7 Data collection

1 Space group P41212 P41212 P41212 Cell dimensions a, b, c (Å) 400.5, 400.5, 175.4 402.6, 402.6, 174.9 402.5, 402.5, 174.8 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å)2 50-3.45 (3.54-3.45) 50-3.3 (3.39-3.30) 50-3.75 (3.85-3.75) Rmerge 0.112 (0.705) 0.102 (0.791) 0.144 (0.858) I / σI 16.3(2.3) 18.8 (2.02) 15.5 (1.99) Completeness (%) 99.4 (100.0) 99.9 (100.0) 99.9 (100.0) Redundancy 11.3 (11.2) 11.3 (11.4) 11.3 (11.4) Refinement Resolution (Å) 20-3.45 (3.49-3.45) 50-3.3 (3.34-3.30) 50-3.75 (3.79-3.75) No. reflections 184776 (5734) 213574 (6618) 146005/4584 Rwork / Rfree 0.152/0.192 (0.251/0.303) 0.154/0.192

(0.260/0.314) 0.148/0.200 (0.234/0.296)

No. atoms Protein 19154 19129 19129 RNA 33017 33097 33254 Ligand/Ion/Water 894 1324 1276 B-factors Protein 123.6 127.5 149.5 RNA 113.9 115.2 141.7 Ligand/Ion/Water 123.1 133.7 152.7 R.m.s deviations Bond lengths (Å) 0.005 0.006 0.014 Bond angles (°) 0.87 0.93 1.63 Ramachandran plot Favored (%) 1852 (89.6) 1844 (89.4) 1794 (87.0) Allowed (%) 193 (9.3) 190 (9.2) 235 (11.4) Generously allowed (%) 21 (1.0) 26 (1.3) 32 (1.6) Disallowed (%) 0 (0.0) 2 (0.1) 1 (0.0) 1. One crystal used for each dataset. 2. Highest resolution shell is shown in parenthesis.

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897-902 (2001). 49. Jenner, L.B., Demeshkina, N., Yusupova, G. & Yusupov, M. Structural aspects of messenger

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