1. Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions
by Jiansen Jiang, Henry Chan, Darian D. Cash, Edward J. Miracco, Rachel R. Ogorzalek Loo, Heather E. Upton, Duilio Cascio, Reid O’Brien Johnson, Kathleen Collins, Joseph A. Loo, Z. Hong Zhou, and Juli Feigon
Science
Volume 350(6260):aab4070
October 30, 2015
Published by AAAS

2. Two views of the Tetrahymena telomerase holoenzyme.
Two views of the Tetrahymena telomerase holoenzyme. The top left image shows a front view of pseudoatomic models as spacefill superimposed on the cryo-EM map. Models of protein domains connected by flexible linkers and not visible in the cryo-EM map are illustrated as ribbons. The bottom right image shows a back-view schematic with the telomerase-bound telomeric G strand connected to a chromosome via double-stranded telomere DNA. Teb1AB domains are presumed to be ordered when DNA bound.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

3. Fig. 1 Cryo-EM reconstructions of Tetrahymena telomerase holoenzyme.
Cryo-EM reconstructions of Tetrahymena telomerase holoenzyme. (A) Front view of the 9.4 Å cryo-EM map, with the catalytic core, Teb1C-Teb2N-Teb3 (TEB), p75C-p45N-p19 (CST), and p50N colored in blue, gold, copper, and red, respectively. (B) Front view of the 9.4 Å cryo-EM map (gray surface) with pseudoatomic models of the TERT-TER-p65 catalytic core, Teb1C-Teb2N-Teb3, and p75C-p45N-p19. (C) Side view of the cryo-EM map and pseudoatomic models shown in (B). (D) Back view of the 8.9 Å cryo-EM map colored as in (A). (E) Back view of the 8.9 Å cryo-EM map, showing pseudoatomic models of TERT-TER-p65 and Teb1C-Teb2N-Teb3. (F) Close-up views of fitting of TER helical domains PK, SL2, S1, and SL4 and TEN domain into the 8.9 Å cryo-EM map. TERT domains are TEN (cyan), TRBD (dark blue), RT (violet, with IFD labeled in violet), and CTE (light blue). Other proteins and TER are colored individually.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

4. Fig. 2 Structure of the TERT-TER-p65 catalytic core.
Structure of the TERT-TER-p65 catalytic core. (A) Model structure of TERT, showing the putative IFD (light violet), active-site catalytic triad residues (red), linker (dotted blue line), and TER TRE. (B) Secondary structure of TER. Locations of CTE and TRBD on TER are indicated with dotted lines. (C) Pseudoatomic model of TERT ring-TER, showing TBE-template-TRE and L4 on TERT, viewed from the active-site side of TERT. (D) Front view of the 8.9 Å cryo-EM map, showing a pseudoatomic model of TER. (E) Front view of the pseudoatomic model TERT-TER-p65 catalytic core. The putative location of IFD is shown as violet oval. (F) A region of the 8.9 Å cryo-EM map and pseudoatomic model showing L4 at the interface of TRBD (helix α8) and CTE (helix α22a, residues 975 to 983). The numbering of Tetrahymena TERT secondary structure elements follows that of the Tribolium TERT structure.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

5. Fig. 3 Identification of two previously unknown holoenzyme proteins, Teb2 and Teb3.
Identification of two previously unknown holoenzyme proteins, Teb2 and Teb3. Shown are two views of the knob and Teb1C region of the 8.9 Å cryo-EM map with a model of Teb1C-Teb2N-Teb3 based on fitting of RPA70C-RPA32N-RPA14 into the cryo-EM map, followed by replacement of RPA70C with Teb1C, except for the RPA70C C-terminal α helix. The TEN domain is shown in cyan. The three-helix bundle between the C-terminal residues of Teb1C, Teb2N, and Teb3 (modeled from RPA) is highlighted with dashed lines. The inset shows the corresponding back view of the holoenzyme for reference.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

6. Fig. 4 Subunit interactions between p50, TEN, IFD, Teb1C, Teb2N, and Teb3.
Subunit interactions between p50, TEN, IFD, Teb1C, Teb2N, and Teb3. (A) Region of the 8.9 Å cryo-EM map showing density linking p50N, TEN, IFD, and TEB. The inset shows a schematic of the interactions. Dotted lines represent inferred interactions from the cryo-EM density and atomic models fitted to the cryo-EM map. (B) Interactions between p50N (red, cryo-EM density), TEN, Teb1C, and Teb2N. The TEN residue K90 side chain corresponding to the human TEN domain residue K78 that interacts with TPP1, R137, and the β3-β4 hairpin (residues 112 to 120) is highlighted in violet. (C) Interactions between TEN and Teb1C. Teb1C F590/F648 residues (orange stick) that together abrogate the Teb1C-TEN interaction and the TEN β3-β4 hairpin (violet) are indicated. TEN residues 122 to 127, disordered in the crystal structure, are represented by dotted lines in (B) and (C). (D) Interactions between TEN and Teb2N. TEN residues 77 to 87, disordered in the crystal structure, are shown as a dashed line. Pseudoatomic models of TEN, Teb1C, and Teb2N interactions are based on crystal structures of TEN, Teb1C, and RPA32N-RPA14 fit into the 8.9 Å cryo-EM map. (E and F) In vitro reconstitution telomerase activity assays for the effect of (E) TEB proteins and (F) TEN domain mutations on the catalytic core assembled with p50N30. TEN domain mutations were tested without (lanes 1 to 4) and with (lanes 5 to 8) TEB. WT, wild type; RC, recovery control.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

7. Fig. 5 Identification of p75-p45-p19 as a Tetrahymena CST complex.
Identification of p75-p45-p19 as a Tetrahymena CST complex. (A) Crystal structure of p19, an OB fold. (B) Model of p75C-p45N-p19 based on fitting of RPA70C-RPA32N-RPA14 into the 9.4 Å cryo-EM map, followed by replacement of RPA14 by p19, except for the RPA14 C-terminal helix. p19 α2 and α3 account for the density at the end of the tip. Gold and orange arrowheads point to the locations of the p19 and p75 C termini, respectively, previously determined by Fab labeling in negative-stain EM (29). The three-helix bundle between the C-terminal residues of p75, p45N, and p19 (modeled from RPA) is illustrated with dashed lines. (C) Crystal structure of p45C, a WH-WH domain. β5 is domain-swapped from a neighboring protein in the crystal lattice. (D) (Top) Negative-stain EM images of four typical p45-Fab–labeled telomerase holoenzyme particles, showing a cluster of 3 Fab bound to the C terminus of p45 at various locations near the holoenzyme. The side length of each image box is 44 nm. (Bottom) Corresponding outlines of telomerase and Fabs are shown in black and blue, respectively. Red dots indicate the p45C (attached to 3 Fab) and p45N (on telomerase) domains; dotted lines represent the linker.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

8. Fig. 6 Telomere ssDNA exits from the backside of the Tetrahymena telomerase holoenzyme.
Telomere ssDNA exits from the backside of the Tetrahymena telomerase holoenzyme. (A) Negative-stain EM class averages of the telomerase holoenzyme dimerized by primer-biotin-streptavidin-biotin-primer. The side length of each image box is 42 nm. (B and C) Random conical tilt (RCT) reconstruction of dimeric telomerase holoenzymes. One of two telomerase holoenzymes in (C) shows uninterpretable features due to flexible positioning of the two holoenzymes relative to each other. (D) A 9.4 Å cryo-EM map of the telomerase holoenzyme (gray surface) and the position of primer-attached streptavidin (black dashed circle), as identified by negative-stain EM RCT reconstruction in (B) and (C). Teb1B (purple) and ssDNA (green) exiting through Teb1C are modeled by fitting the crystal structure of the RPA:ssDNA complex (PDB ID 4GNX).
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS

9. Fig. 7 Schematics of the complete Tetrahymena telomerase holoenzyme and DNA exit path.
Schematics of the complete Tetrahymena telomerase holoenzyme and DNA exit path. (A) Arrangement of the subunits and domains of the Tetrahymena telomerase catalytic core, TEB, and CST complexes tethered to p50, shown as front view. Domains connected by flexible linkers and not seen in the cryo-EM map are shown with oval outlines. (B) Arrangement of the holoenzyme with proposed path of telomere ssDNA, shown as back view. Teb1AB domains are presumed to be ordered when DNA-bound.
Jiansen Jiang et al. Science 2015;350:aab4070
Published by AAAS