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0 TIAB Cryo-EM structure of substrate-bound human telomerase holoenzyme Summary Telomerase adds telomeric r 1 1197 310 show
1 Body Linear eukaryotic chromosomes are capped by telomeric repeat tracts that recruit the telomere-bindin 1 4717 0 show
2 Different activities for distinct structural states of purified human telomerase We reconstituted human telomerase holoenzyme by transient transfection of HEK 293T cells with vector 1 1812 0 show
3 Overall architecture and flexibility of human telomerase We prepared ΔTCAB1 telomerase with a hTR CAB box mutant that disrupted TCAB1 binding25,26 and TERT-h 1 1799 0 show
4 Subunit distribution across the two lobes of human telomerase The bilobal human telomerase includes one TERT molecule and one hTR (Fig. 2a and b), in agreement wi 1 3710 0 show
5 The catalytic core The t/PK and CR4/5 domains of hTR wrap around TERT to form the catalytic core (Fig. 4a). In turn, th 1 3843 0 show
6 The H/ACA RNP and human disease mutations Our structure offers the first glimpse of the subunit architecture of an intact eukaryotic H/ACA RNP 1 3600 0 show
7 Conclusions The structure presented here provides the first architectural characterization of a telomerase holoe 1 426 0 show
8 Methods Telomerase reconstitution Human telomerase was reconstituted in cells with the TERT expression plasm 1 1610 0 show
9 Methods Telomerase purification The prepared whole-cell extract (from 120 plates of adherent cells or 6L of 1 1572 0 show
10 Methods Immunoblotting The purified telomerase samples (E1 and E2) from the CHAPS lysis protocol were resolv 1 1088 0 show
11 Methods Direct telomerase activity assays Direct primer extension assays were performed using 1 μl of sample 1 749 0 show
12 Methods HCT116 cell culture and genome editing HCT116 cells were cultured in DMEM with GlutaMax (ThermoFishe 1 922 0 show
13 Methods Telomeric repeat amplification assays Cell extract was prepared using the HLB method as described ab 1 1415 0 show
14 Methods Southern blotting Genomic DNA preparation was conducted as previously described28. Four-8 ug of DNA 1 804 0 show
15 Methods Negative-stain electron microscopy and data processing For negative-stain grid preparation, 4 μL of 1 2577 0 show
16 Methods Cryo-EM electron microscopy For cryo-EM analysis, 3 μl of the purified telomerase was applied to C-f 1 1210 0 show
17 Methods Image processing Most steps of data processing were done in RELION 2.0 (ref. 64) unless otherwise st 1 5290 0 show
18 Methods Model fitting Initial fitting of all X-ray structures, NMR ensembles and homology models was perform 1 2444 0 show
19 Methods Map and model visualization Maps were visualized in Chimera72 and all model illustrations were prepa 1 150 0 show
21 Caption-Extended Data Figure 1 | Protein identification by immunoblotting, enriching active telomerase, substrate pre-binding, and co 1 2027 0 show
22 Caption-Extended Data Figure 2 | Cellular function of the tagged TERT used for structural analysis. a, Western blot detection of ZZ-S 1 2034 0 show
23 Caption-Extended Data Figure 3 | Image processing procedures. a, Representative raw micrograph. We collected a total of 11,654 microg 1 253 0 show
24 Caption-Extended Data Figure 4 | Resolution estimation and analysis of the flexibility of the complex. a, Representative 2D class ave 1 1329 0 show
25 Caption-Extended Data Figure 5 | Fittings of proteins and RNA into the cryo-EM map. a-d, Domains of TERT. a, The TEN domain from Tetr 1 695 0 show
26 Caption-Extended Data Figure 6 | Sequence alignment of TERT with secondary structure assignments based on known structures. a, Sequen 1 834 0 show
27 Caption-Extended Data Figure 7 | Selected protein-protein and protein-RNA interactions in telomerase holoenzyme and comparisons betwe 1 2103 0 show
28 Caption-Extended Data Figure 8 | Sequence alignments of H/ACA proteins with secondary structure assignments based on known structures 1 752 0 show
29 Caption-Extended Data Table 1 | Mass spectrometry analysis of the purified telomerase sample. Proteins are ranked according to their 1 361 0 show
30 Caption-Extended Data Table 2 | Summary of cryo-EM data collection and modelling of protein and RNA subunits 1 76 0 show
31 Caption-Figure 1 | Telomerase holoenzyme reconstitution and characterization. a, Domain architecture of TERT (top) and 1 1003 0 show
32 Caption-Figure 2 | Cryo-EM structure of the substrate-bound human telomerase holoenzyme. a, Schematic of subunit arrang 1 297 0 show
33 Caption-Figure 3 | hTR structure in human telomerase holoenzyme. a, Schematic of secondary structure of hTR, with domai 1 397 0 show
34 Caption-Figure 4 | Structure of the catalytic core. a, Front (left panel) and back (right panel) views of the catalytic 1 1302 0 show
35 Caption-Figure 5 | The H/ACA domain and disease mutations. a, Front and back views of the H/ACA lobe within the human t 1 773 0 show