dr ss jan20 supplementary images - nature · structure with known handedness (a tubular assembly of...

Supplementary Information

Structure of Active, Dimeric Human Telomerase

Anselm Sauerwald, Sara Sandin, Gaël Cristofari, Sjors H.W. Scheres, Joachim Lingner, and

Daniela Rhodes

Nature Structural & Molecular Biology: doi:10.1038/nsmb.2530

Table S1: Composition of purified and active human telomerase .

Unique hTERT, hNop10, and human dyskerin peptides sequenced by nanoLC-MS/MS.


Figure S1: Analysis of purified active human telomerase. a, SDS-PAGE analysis of the telomerase was on 4-20% acrylamide gel and the gel silver-stained: Telomerase captured on IgG-Sepharose via a protein A-tag present in the TERT subunit and eluted by TEV cleavage (middle lane). Telomerase further purified using a heparin column (right lane). Size markers are shown in the left lane. b, EMSA of a twofold dilution series of IgG-Sepharose/Heparin purified human telomerase bound to 32P labeled (TTAGGG)3 telomeric DNA. As control, oligo DNA was incubated without telomerase. The analysis was on a 6.7% acrylamide gel and visualized by autoradiography. c, Molecular weight estimation of active human telomerase enzyme complex. Profile of the purified telomerase bound to 5’-[32P] (TTAGGG)2-3’ complex analysed by sedimentation on a 5 to 30 % sucrose gradient. Green bars represent the pmol amount of 5’-[32P]-(TTAGGG)2-3’. The black line shows the sedimentation profile of thyroglobulin which has a MW=669 kDa.

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Figure S2: Correlation of telomerase activity with wild-type hTERT(WT) and catalytic dead hTERT(DN) expression hTR(WT) was transiently co-expressed with either ZZ 3xFlag hTERT(WT), 13xMyc hTERT (WT), or the catalytically dead mutant ZZ 3xFlag hTERT(DN) as described in Online Methods. a, TERT expression were determined using Western blot analysis of TEV treated whole cell lysates with an anti C-terminal hTERT antibody (Lanes 2, 3 and 4) . Whole cell lysate of untransfected HEK 293T cells was used as an expression control (Lane 1) b, Telomerase activity was analyzed using the Telospot assay of whole cell lysates. c, TERT expression and telomerase activity were correlated by Western blot analysis of a 2-fold serial dilution of TEV treated whole cell lysate (Lanes 2 to 5) and of immunoaffinity purified and TEV released complexes (Lanes 7 and 8) with anti C-terminal hTERT antibody. Whole cell lysate of untransfected HEK 293T cells was used as an expression control (Lanes 1 and 6). hTERT expression levels were measured by quantitative western analysis and shown in arbitrary units. d, Telospot telomerase activity assay of the whole cell lysate serial dilution (Lanes 2 to 5) and of immunoaffinity purified and TEV released complexes (Lanes 7and 8). The quantified telomerase activity shown in both b and d is given as a multiple of the activity of untransfected HEK293T whole cell lysate.

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Figure S3: Single particle refinement of the telomerase dimer. Electron tomography, combined with reference-free sub-tomogram averaging was used to calculate an initial low-resolution 3D reconstruction of the telomerase dimer (a). Refinement of this structure against a data set of 20,127 single particle images (b) gave rise to indications of structural heterogeneity in the data. Distinct conformations were then classified by three-dimensional maximum-likelihood analysis in Fourier space (MLF3D), which was used to calculate reconstructions for four subsets of the data (c-f). Images included in each of these classes are 3659(c), 2631(d), 2608(e) and 5519(f).

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Figure S4: Tilt pair validation and determination of the absolute hand. Scatter plot of the relative transformations between independently assigned orientations of individual dimer projections. The particles used in this analysis were part of a single tilt series and ranged in nominal tilt -40 to 0 degrees. The particle with a nominal tilt angle of -2 degrees was used as a reference to calculate all relative tilt transformations. A vector from the origin to any point in the plot corresponds to the direction of the relative tilt axis, and its length is the tilt angle. The colour of each point represents to what extent the relative tilt-axis lies in the actual plane of tilting: blue points have an in-plane tilt axis; purple to red points have an increasing out-of-plane tilt axis. The observation that most points are blue and that they all lie on an approximate line is as expected for a single tilt series, which validates the overall correctness of the reference structure. A similar plot for a tilt series (collected on the same microscope) on a structure with known handedness (a tubular assembly of acetyl choline receptors) indicates that the handedness of the structure is correct.


Figure S5: Docking and subunit assignment of the open monomer density map. a A continuous tubular density with a diameter of 25-30 Å is located opposite the exit site of the 5’ end of the DNA (red). This density has the dimensions expected of an RNA helix as shown by docking a modeled ideal RNA helix (in yellow) into the EM density. TERT-like protein subunit (blue) in complex with a DNA strand in the catalytic site (red) docked into the three-dimensional map of the telomerase monomer (transparent surface representation). The views are related by a 90º rotation around the vertical axis. b X-ray crystallographic structure of the TERT-like protein subunit (blue) superimposed with a low pass filtered density map of the same structure (grey surface representation). The density map was generated and filtered to 23 Å in Chimera.

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Structure of Active, Dimeric Human Telomerase

Supplementary Note

EM sample preparation and imaging. Continuous carbon-coated grids were freshly

prepared and glow-discharged before use. 13 µl of telomerase sample (8-10 nM) were

deposited on the grid for 15-30 minutes, blotted with filter paper and negatively

stained with 2 drops of 1-2% (w/v) uranyl acetate solution. A homogenous sample

preparation for EM was obtained using these conditions although the particle

concentration was low (30-100 particles/image). Single particle EM data were

collected on a FEI CM12 transmission electron microscope, operated at 120 keV and

liquid nitrogen temperature. The nominal magnification was 42,000× (calibrated

magnification, 42,550×). Micrographs were recorded on Kodak SO-163 film with an

electron dose of 10-12 e/Å2 and a defocus of 1.0–1.5 µm. The films were developed

in Kodak developer at full strength for 12 min and digitized with a Zeiss SCAI

scanner using a step size of 7 µm. The micrographs were compressed x4 giving a final

object pixel size of 6.6Å/pixel. Figures 1d and colloidal gold data were recorded on a

2k TVIPS CCD camera with an object pixel size of 3.25 Å. Single axis tilt series were

recorded with SerialEM1 on a FEI Tecnai G2 Polara microscope at 300keV and 3.5

µm underfocus. The specimen was tilted ±60° or ±65° and images were recorded

every second degree on a 2k TVIPS CCD detector. The electron dose per image was

10 e/Å2 and the pixel size was 5.65 Å at the specimen level.

Particle selection and 2D class averaging. A data set of 26,361 telomerase particles

bound to oligonucleotide 5’-(TTAGGG)2- 3’ were selected manually with Ximdisp2

using a box size of 78x78 pixels. The image stack was normalised and sorted by


statistics in Xmipp3, and 20,127 images were selected for further analysis. A

maximum likelihood target function in Fourier space (MLF2D4) was used for multi-

reference alignment and 2D classification.

Initial low-resolution 3D reconstruction by electron tomography. Electron

tomography was used to calculate a low-resolution reference model. Single axis tilt

series were compressed x2 giving a pixel size of 11.3 Å at the specimen level. The

micrographs were coarsely aligned by cross-correlation and reconstructed by filtered

back-projection in IMOD5. An iterative refinement procedure was used to refine the

alignment parameters and the reconstruction in EMTIAR6. 50 evenly stained sub-

tomograms corresponding to individual telomerase particles were manually selected

in Bsoft7 and extracted using a box size of 40x40x40 voxels. An average 3D

reconstruction was calculated by missing-wedge weighted, reference-free alignment

of sub-tomograms in Xmipp8. 13 sub-tomograms with the highest cross correlation to

the average (Fig. 1f and fig. S3a) were included in the final sub-tomogram average.

Map refinement by single particle electron microscopy. Initial single-particle

refinement was performed in EMAN29 using the sub-tomogram average (fig. S3a) as

a starting reference. The reference model was initially filtered to 80 Å resolution and

refined (fig. S3b) by 20 rounds of multi-reference alignment, classification by

multivariate statistical analysis and angular assignment by projection matching. Class

averages with 6 degree angular spacing were calculated using 6 averaging iterations.

The 3D structure was calculated at 35 Å resolution by direct Fourier inversion. To

analyse structural variability in the telomerase dimer we used 3D-classification by

maximum-likelihood in Fourier space (MLF3D4). After 25 rounds of non-supervised

angular refinement against five reference models, four homogenous subsets of images

were selected for further refinement (a fifth class was interpreted as an accumulation


of particles of low quality). The selected classes contained 2608, 2631, 3659 and 5519

particles respectively. Subsequent refinement in EMAN2 (as described above) yielded

reconstructions with resolutions in the range of 30-40 Å (fig. S3c-f).

Map validation and absolute hand determination. The absolute hand and the

overall correctness of the dimer structure in Figure 3f were assessed using the tilt-pair

validation method10. From one of the tilt series that was used to calculate electron

tomograms for our reference-free initial model calculation, we selected the individual

projections of one of the dimer particles (selection of other particles gave similar

results). These single-particle projections were then aligned independently against

reference projections of the dimer structure. Analysis of the relative tilt angles

between these images (fig. S4) validated the structure, while comparison with an

identical analysis for a tilt series that was collected on the same microscope on a

sample of known handedness (helical assemblies of acetyl choline receptors) served

to determine its absolute hand.

Independent monomer refinement and reconstruction of composite dimers. From

2D class averages of telomerase dimer side-views, we calculated the centre of each

monomer in the raw image stack; 9,380 sub-images were selected of the open

monomer and the closed monomer (4,690 each), and extracted using a box size of

34x34 pixels. These sub-images were used for independent monomer refinement,

using a two-reference model procedure, in Xmipp (Fig. 4-5). To re-combine the

monomers into composite dimers, reference-free 2D class averages (Fig. 4c top

panel), were used to determine the orientation of the open and closed monomers

(calculated as the average orientation of all sub-images that were included in the 2D

class average). This analysis provided a reconstruction of the relative orientation of

the independely refined monomers (Fig. 4c middle panel). Since the analysis was


carried out using 2D class averages of side views, the Z-height of the monomers were

adjusted by hand and compared to the previously refined intact dimer (Fig. 3f). Visual

inspection shows that projections of the assembled composite dimer (Fig. 4c, lower

panel) agreed well with the 2D class averages (Fig. 4c top panel).

Colloidal gold labeling of telomerase domains. Two types of 5 nm colloidal gold,

coated with either streptavidin (prepared as described 11) or Ni-NTA (Nanoprobes,

Inc Yaphank, NY, USA), was used to identify the presence and location of the

telomeric G-overhang and the TERT subunit respectively. The binding integrity of the

monovalent streptavidin gold complex to 5’-[32P]/biotin 5’-[32P]-(TTAGGG)2-3’ -3’

was analysed by EMSA. After GraFix purification of the telomerase (as described

above), streptavidin coated gold was added in a stochiometric ratio of 2:1 to

telomerase bound to 5’- Biotin dT-(TTAGGG)2- 3’ and incubated for 30 minutes on

ice before preparing EM grids (Fig. 1g). To map the location of the TERT subunit,

telomerase N-terminal 6xHis-tag (ZZ_(TEV)_6xHis_5x(AAAKE) ~37Å long alpha

helix_3x_Flag_hTERT) was incubated with Ni-NTA coated gold for 3 hours at room

temperature (Fig. 5d). We found more telomerase particles in complex with

streptavidin coated gold (87%) compared to Ni-NTA coated gold (16%), which is

likely to reflect the high affinity of streptavidin-biotin (not shown).

Model fitting and density visualization. The crystal structure of the TERT protein

subunit (pdb code: 3KYL) was docked into the EM density of the monomeric subunit

using automatic docking procedures in Chimera12. This program was also used for 3D

density visualization and generating a 23 Å density map of the TERT subunit (Fig.

S5b).


1 Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36-51, doi:10.1016/j.jsb.2005.07.007 (2005).

2 Smith, J. M. Ximdisp--A visualization tool to aid structure determination from electron microscope images. J Struct Biol 125, 223-228, doi:10.1006/jsbi.1998.4073 (1999).

3 Sorzano, C. O. et al. XMIPP: a new generation of an open-source image processing package for electron microscopy. J Struct Biol 148, 194-204, doi:10.1016/j.jsb.2004.06.006 (2004).

4 Scheres, S. H. et al. Modeling experimental image formation for likelihood-based classification of electron microscopy data. Structure 15, 1167-1177 (2007).

5 Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 71-76 (1996).

6 Chen, H., Hughes, D. D., Chan, T. A., Sedat, J. W. & Agard, D. A. IVE (Image Visualization Environment): a software platform for all three-dimensional microscopy applications. J Struct Biol 116, 56-60 (1996).

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8 Scheres, S. H., Melero, R., Valle, M. & Carazo, J. M. Averaging of electron subtomograms and random conical tilt reconstructions through likelihood optimization. Structure 17, 1563-1572 (2009).

9 Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157, 38-46, doi:10.1016/j.jsb.2006.05.009 (2007).

10 Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J Mol Biol 333, 721-745 (2003).

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dr ss jan20 supplementary images - nature · structure with known handedness (a tubular assembly of...

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