1. Volume 10, Issue 1, Pages 117-128 (July 2002)
The Structures of Four Macrolide Antibiotics Bound to the Large Ribosomal Subunit 
Jeffrey L. Hansen, Joseph A. Ippolito, Nenad Ban, Poul Nissen, Peter B. Moore, Thomas A. Steitz 
Molecular Cell 
Volume 10, Issue 1, Pages (July 2002)
DOI: /S (02)

2. Figure 1
Chemical Structures of the Macrolides, Tylosin, Carbomycin A, Spiramycin, Azithromycin, and Erythromycin
Atoms in these figures and in the Protein Data Bank coordinate files (1K8A, 1K9M, 1M1K, and 1KD1) are named according to Paesen et al. (1995), with the numbering of the atoms of the lactone ring starting at the ester bond. Oxygen atoms are numbered according to the adjacent carbon atoms, and sugar atom numbers are modified by suffixes A, B, or C to distinguish mycaminose, mycarose, and any additional sugar, respectively.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
A Secondary Structure Diagram of the Peptidyl Transferase Loop and a Loop from Domain II of 23S rRNA Along with the Sequence Numbers of Nucleotides Mentioned in the Text
The first number is the Haloarcula marismortui sequence number and the second number (in parentheses) corresponds to the sequence number in E. coli 23S rRNA.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Electron Density Maps of the Macrolide Binding Site with Drugs Bound
(A and B) Electron density map calculated using 2Fo-Fc as coefficients showing the position of azithromycin (A) or tylosin (B). Electron density corresponding to the lactone ring is donut shaped, with the expected hole clearly establishing the position of the macrocyclic ring. These maps are unbiased, since the coordinates of the macrolide were not included in either the refinement of the ribosome coordinates or in the calculation of the phases used in the map calculation.
(C) Covalent bond. A 2Fo-Fc Fourier map calculated with carbomycin A excluded from the model and the phasing; continuous electron density (black net) connecting the macrolide (orange) to A2103 (2062) (green), consistent with the formation of a carbinolamine bond (near tip of black arrow). The resulting hydroxyl group (red sphere) is out of plane with respect to the base of A2103 and therefore fits the bulge in electron density, consistent with the tetrahedral geometry of a carbinolamine bond, and inconsistent with the planar geometry of an exocyclic Schiff's base. An acetyl group at C3 of carbomycin A forms a hydrogen bond (dotted line) with the newly available hydroxyl group which was formed by reduction of the aldehyde. The model coordinates that were used in all steps of the refinement and for this map calculation contained neither the antibiotic nor the extended conformation of A2103.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Global Views of the Macrolide Binding Site Showing How They Block the Peptide Exit Tunnel
(A) Macrolide binding site. The top of the ribosome is rotated backward from the crown view so that the peptide exit tunnel (gray) with modeled peptide (orange ribbon) can be seen when the front half of the ribosome is removed. Ribosomal RNA is white and ribosomal proteins are light blue space-filling representation. The E-site tRNA (red sticks), A-site tRNA (purple sticks), and P-site tRNA (orange sticks) have been positioned using the coordinates from the 70S ribosome complex after superposition of corresponding 50S subunit atoms. Black arrows point to an opening between the peptidyl transferase center and the peptide exit tunnel where macrolides (red) bind. The green atoms are bases that interact with the macrolides.
(B) Constriction. An alternate view up the exit tunnel, through the constriction, shows P-site (orange sticks) and A-site (purple sticks) substrates bound to the active site (J.L.H., M. Schmeing, P.B.M., and T.A.S, unpublished data).
(C) Blockage. The opening shown in (B) is occluded by both the presence of the macrolide (red) and the extended conformation of A2103 (2062) (green). In both Figures 3C and 3D, A2103 (2062) is shown in the extended conformation, because binding of either macrolides or P-site substrates results in the extended conformation.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
Comparison of the Interactions of Different Macrolides with the Ribosome
(A) Carbomycin (red), tylosin (orange), spiramycin (yellow), and azithromycin (blue) bind the ribosome in an almost identical fashion and cover G2099 (A2058) and A2100 (2059) (green spheres). The lactone ring is extended further into the tunnel by mycinose on tylosin and forosamine on spiramycin. The disaccharide moiety extends the 16-membered macrolides in the opposite direction toward the catalytic center. Upon 16-membered macrolide binding (but not azithromycin), the base of A2103 (2062) (dark green) moves (curved white line) from its location against the wall of the exit tunnel to an extended conformation (light green sticks) and forms a covalent bond with the macrolide (orange sticks). The isobutyrate group of carbomycin A (red) reaches into the tRNA A-site (dark blue and purple spheres). The mycinose moiety of tylosin (orange) contacts protein L22. The forosamine moiety of spiramycin (yellow) contacts L4. The cladinose sugar of azithromycin binds in a fourth sugar binding pocket. These three macrolides were aligned by least squares superimposition of the phosphates of ribosomal RNA.
(B) Alignment of erythromycin (white) bound to the D. radiodurans large subunit (Schlünzen et al., 2001) with azithromycin (blue) bound to the H. marismortui large subunit.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6 Macrolide Binding and Resistance Mutations
(A) Lactone ring. A hydrophobic binding site that contacts the hydrophobic face of the lactone ring (orange) is provided by the RNA bases of G2099 (A2058) and A2100 (A2059) (green), which splay apart, and base pair C2098-G2646 (G2057-C2611) (gray sticks). H bonds are shown as dotted lines. N2 (blue sphere) of G2099 (A2058) at the center of these hydrophobic interactions appears to be destabilizing to macrolide binding (red arrows).
(B) Mycinose sugar of tylosin. The mycinose sugar (orange sticks) of tylosin interacts with domain II of ribosomal RNA (gray sticks) through hydrophobic van der Waals contacts hydrogen bonding (black dotted lines). This structure was modified by modeling in a methyl group bonded to N1 of G841 (A748). Resistance conferred by that modification is apparently caused by the sterically blocking of part of the the mycinose binding pocket and by loss of the hydrogen bond between the N6 of the A and the sugar O2C.
(C) Mycaminose sugar and G2099 (A2058). The O2A of mycaminose forms a hydrogen bond (black dots) with N1 of G2099 (A2058). O6 (red sphere) of G2099 (A2058) (green) is located adjacent to the mycaminose sugar within 3.6 Å of its dimethyl amine group.
(D) In this model, the structure from Figure 6C was changed by replacing G2099 (A2058) with dimethyl adenine, a modification that confers resistance to macrolides. The resistance is evidently due to the methyl groups sterically clashing with the methyl amine group and the O2A of the mycaminose sugar. This would not allow the sugar to bind with the same position and orientation and would interfere with the one hydrogen bond between O2A and N1 of nucleotide 2099 (dotted line).
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
The Correlation between Macrolide Structures and Their Effect on Protein Synthesis
Carbomycin A in stick representation with the three moieties attached to the C5 of carbomycin lactone ring, mycaminose (orange), mycarose (yellow), and isobutyrate (gray) in order to highlight the inverse relationship between the length of the C5 branch of a macrolide and the length of an oligopeptide (brown α-carbon spheres) that can be synthesized in the presence of the macrolide. The isobutyrate group of carbomycin reaches the peptidyl transferase center and occupies the A-site amino-acid binding pocket, which would prevent binding of an A-site substrate (purple α-carbon sphere number 1) and prevents formation of even a dipeptide on a P-site substrate (brown α-carbon spheres number 1 and 2). Tylosin extends to the mycarose sugar (yellow) and allows only dipeptide formation. Azithromycin (and erythromycin) which have only a monosaccharide extension from C5 of the lactone ring (orange) allow formation of up to tetra-peptides.
Molecular Cell  , DOI: ( /S (02) )