Cryo-EM structure of the human CST–Polα/primase complex in a recruitment state

The CST–Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST–Polα/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus. Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST–Polα/primase complex map to protein–protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.

184-398 aa) in that region (Fig. 1c) and, because ssDNA competes with STN1 C for that binding site 13 , we retained the 4 nt of ssDNA from the previously determined CST structure 13 in the model we docked into our map (Fig. 1c).
After initial rigid-body docking followed by flexible fitting and refinement, the overall conformations of the two subcomplexes did not show major changes from their structures in isolation ( Fig. 1c and Extended Data Fig. 3f). In the complex, Polα/primase remains in the occluded state 17 , in which the POLA2 subunit is blocking entry of DNA into the active site of POLA1 (Fig. 1c). This finding is consistent with reported results showing that cross-linking preferentially stabilizes the more compact, occluded state compared with the flexible, extended state of the enzyme 21,22 . Thus, we conclude that our structure likely captures a recruitment state of the complex that forms prior to active RNA and DNA synthesis by Polα/primase. Structural and evolutionary analysis of the CTC1-POLA1 interface. The primary interaction interface observed in our structure occurs between the C-terminal domain of POLA1 (POLA1 CTD , 1,265-1,462 aa) and the N-terminal OB folds of CTC1 (Figs. 1c and 2a). The resolution is limiting for rigorous analysis of amino acid interactions, and analysis of surface electrostatic potential suggests that this interface is not driven by a dominant hydrophobic or charged interaction, but rather by shape complementarity of the two proteins, burying 1,250 Å 2 of solvent-accessible surface area ( Fig. 2a and Extended Data Fig. 4a,b).
We identified a CTC1-recognition loop (CRL, 1400-1424 aa) in the POLA1 CTD that is shifted relative to its position in the apo structure 17 to contact CTC1 (Fig. 2a). Sequence conservation analysis of the interface revealed that both CTC1 and the interacting region on the POLA1 CTD have low conservation at the primary sequence level (Extended Data Figs. 4c and 5a). However, the CRL is identifiable by an insertion of uniform length in metazoans, and we find that, when modeled using predictions from AlphaFold 2 (refs. 19,20 ), the CRL is structurally conserved in metazoans. Furthermore, we find that the presence of a CRL feature correlates with an expansion of metazoan CTC1 to contain the N-terminal OB folds that interact with the CRL. In unicellular eukaryotes, this loop diverges greatly between species and can be either shorter (for example, in Tetrahymena thermophila and Schizosaccharomyces pombe) or longer (for example, in Saccharomyces cerevisiae) and adopts a different predicted structure compared with that in the metazoan CRL ( Fig. 2b and Extended Data Fig. 5b).
To assess the involvement of the CRL in CST binding, we generated human POLA1 CTD constructs with the wild-type CRL (CRL WT , 1265-1462 aa), with the CRL swapped for a GGSGGS-linker (CRL GGSGGS , 1265-1402-GGSGGS-1423-1462 aa), or with the CRL swapped for the S. pombe short loop (CRL S. pombe , 1265-1399-QTTTGAT-1425-1462 aa) (Extended Data Fig. 6a-d). Although the POLA1 CTD constructs ran as heterogeneously sized smears in SDS-PAGE, they compressed to single bands in native PAGE (Extended Data Fig. 6a,b). Sharp symmetric peaks in SEC elution profiles (Extended Data Fig. 6c) and spectrophotometric (Nanodrop) quantification indicated that the protein was pure and free from nucleic acid contamination, respectively. We measured the affinity of the interaction between the POLA1 CTD constructs and fluorescently labeled CST using microscale thermophoresis (MST) (Fig. 2c). Two distinct binding events between the CRL WT protein and CST were observed: the first binding event (1; Fig. 2d) is higher affinity and characterized by a positive change in the normalized fluorescence (Fig. 2d), and the second binding event (2) is lower affinity and characterized by a negative change in the normalized fluorescence (Fig. 2e). We separated the data on the basis of the two inflection points 23 and calculated dissociation constant (K D ) values of ~3.3 μM and ~122 μM for binding events 1 and 2, respectively. In contrast, the CRL GGSGGS and CRL S. pombe mutant proteins displayed binding in only the second event (Fig. 2c,d). For binding event 2, the determined K D values for the CRL GGSGGS and CRL S. pombe proteins were ~96 μM and ~88 μM, respectively.
The CRL and one Zn 2+ -binding domain in POLA1 CTD form the module that fits into a complementary cleft in CTC1. We speculate that the high-affinity binding mode (event 1; Fig. 2d) observed in the MST experiments involves both the CRL and Zn 2+ -binding domain, whereas the lower affinity binding mode (event 2; Fig. 2e) reflects the interaction of the Zn 2+ -binding domain with CTC1, which is CRL-independent. In this second binding mode, CST could rock about the Zn 2+ -binding domain, echoing the hinge-like flexible motion of CST about POLA1 suggested by the cryo-EM data (Extended Data Fig. 3d,e). CTC1 OB-D , an elongated OB fold that shares no homology with known OB folds 13 , forms the major interaction with POLA1 CTD (Fig. 2a and Extended Data Fig. 4). This finding is particularly interesting given the proposal that CST and Polα co-evolved in eukaryotes 24 . Our structure would then capture a metazoan-specific development in this trajectory.
Our structural model also places regions of CTC1 near POLA2 and PRIM2 (Fig. 1c). These two potential interaction sites are not   well resolved in our cryo-EM map, but it is possible that these interactions are weak and/or more transient as CST flexes about the hinge generated by the POLA1 CTD -CTC1 interaction.
CST-Polα/primase maintains a 1:1 stoichiometry. The CST-Polα/primase complex is sterically incompatible with the previously reported ssDNA-bound CST decamer 13 , as it would bind in the center of the ring and sterically clash with neighboring CST subunits ( Fig. 3a). CST has also been shown to dimerize upon ssDNA binding 13 , but two additional lines of evidence suggest that active CST-Polα/primase has a 1:1 stoichiometry. First, we do not observe CST dimers in our 2D class averages (Extended Data Figs. 1 and 2). Second, we characterized the CST-POLA1 N interaction to understand why CST-PP FL did not yield a high-resolution map although PP FL forms a more stable interaction with CST. We reconstituted a native complex of CST and MBP-tagged POLA1 N and analyzed it by negative-stain EM and cross-linking mass spectrometry (CX-MS) (Extended Data Fig. 7). With MBP as a mass label, we localized the N-terminus of POLA1 N to the primary CST dimerization interface (Fig. 3b). CX-MS analysis suggests that POLA1 N binds in multiple modes to CST, which could partially explain the heterogeneity observed with CST-PP FL (Fig. 3c,d). It is possible that POLA1 N binding is restrained in the presence of the full complex, but we observe similar cross-links between CTC1 and POLA1 N in CX-MS analysis of CST-PP FL (Extended Data Fig. 7c). Thus, we conclude that POLA1 N binds heterogeneously to CST in the region of the dimer interface, and the CST-Polα/primase fill-in machinery functions as a 1:1 complex.
Coats plus mutations map to recruitment interfaces. Three CP point mutations (p.A227V, p.V259M, and p.V665G) in CTC1 were previously described to disrupt Polα/primase association with CST 14,25 (Fig. 4a). We mapped these residues onto our structure to investigate the molecular basis of dysfunction caused by these mutations (Fig. 4b). V665 resides on a β-strand of CTC1 OB-D , so it is plausible that a glycine substitution would destabilize the β-sheet and OB fold, disrupting the primary interaction. The mutations at A227 and V259 reside on CTC1 OB-B , which does not contact Polα/ primase in the CST-PP ΔN structure (Fig. 4b). However, one major difference we observe between the cryo-EM maps of CST-PP ΔN and CST-PP FL is the presence of connecting density between POLA2 and the CTC1 N-terminus, which we only observe when POLA1 N is present (Fig. 4c). Because cooperative binding between POLA1 N and POLA2 NTD (1-78 aa, attached by a flexible linker and not visualized in our CST-PP ΔN structure) in other settings has been described 26 , we speculate that this bridging density is a combination of these two termini. Although our resolution is limited, this connection could potentially explain the CP mutations occurring at the CTC1 N-terminus (Fig. 4a-c).

Discussion
In this study, we report a novel structure of the human CST-Polα/ primase fill-in machinery. By chemically cross-linking the complex, we captured a conformational state that reveals how CST can recognize and bind the occluded state of Polα/primase through a newly uncovered interaction between CTC1 and POLA1 CTD . We propose that this interaction is specific to metazoans and occurs during recruitment of Polα/primase to the telomere, prior to the start of RNA/DNA synthesis by the enzyme. Notably, this interface is formed by the N-terminal four OB folds of CTC1, a metazoan expansion of the subunit that further differentiates it from the paralogous RPA large subunit and from CTC1 homologs found in unicellular eukaryotes 13,24 . Our evolutionary conservation analysis identified a complementary species-specific loop in the POLA1 CTD , termed the CRL, that appears to have co-evolved with the expansion in CTC1. Substitution of the CRL with a short loop, either a GGSGGS linker or the orthologous S. pombe sequence, abrogates binding in the high-affinity (K D = ~1-10 μM)  2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 20 2 20 2 2 2 2 20 20 20   binding mode between POLA1 CTD and CST. MST experiments also captured an order-of-magnitude-lower affinity (K D = ~100 μM) binding mode that is CRL-independent. The two binding modes are consistent with our cryo-EM structure, in which POLA1 CTD utilizes both the CRL and a Zn 2+ -binding motif to interact with CTC1.   Further work is needed to delineate the functional role of POLA1 N in regulating the fill-in machinery. We uncovered an interaction between POLA1 N and CST by using a combination of negative-stain EM, CX-MS, and cryo-EM with CST bound to POLA1 N in isolation and additionally bound in the context of PP FL . The CST-PP FL complex associated more robustly than CST-PP ΔN , as inferred from higher CST occupancy in cryo-EM 2D averages and stability during native SEC. However, we observed greater structural heterogeneity in the cryo-EM data for CST-PP FL . The addition of POLA1 N may allow the complex to sample a greater conformational space, interfering with accurate alignment of the particles in the CST-PP FL complex. POLA1 N is responsible for the flexible tethering of Polα/ primase to the replisome via an interaction with AND-1 (refs. 27,28 ), and its extensive yet flexible interaction with CST suggests a potential spatiotemporal regulation of fill-in after telomere replication, where the replisome hands Polα/primase off to a shelterin-bound CST for C-strand synthesis (Fig. 4d).
Finally, our model informs on CP mutations (p.A227V, p.V259M, p.V665G) previously characterized to disrupt CST-Polα/primase association 14,25 . It is unlikely that these single point mutations result in complete loss of function, as such mutations would likely be lethal. Consistent with this framework of mild dysfunction, we observe that the three CP mutations map close to interaction interfaces but are not necessarily responsible for direct interaction with Polα/primase. For example, V665 resides on a β-strand in Ctc1 OB-D that is not directly on the interface, but the glycine substitution could destabilize the OB fold and weaken the interaction. Similarly, the Ctc1 OB-B mutations reside near the bridge observed in the CST-PP FL cryo-EM map. Since those mutants (p.A227V, p.V259M, p.V665G) were characterized 14 , three more CP mutations have been reported in CTC1 OB-B (refs. 29,30 ). Presumably, these would be deleterious in a manner resembling that of p.A227V and p.V259M. Future genetic and functional studies are required to elucidate the precise mechanism by which these mutations cause CP, but our structural results provide a framework for understanding the molecular basis of human disease linked to CST-Polα/primase, its role in telomere maintenance, and its contribution to DSB repair.

online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/ s41594-022-00766-y. TCEP. Dialyzed protein was incubated with end-over-end rotation for 1 h at 4 °C with Ni-NTA resin (Invitrogen) equilibrated with dialysis buffer to rebind the His 6 -Smt3, His 6 -Ulp1, and any uncleaved protein. The flowthrough (containing cleaved POLA1 CTD protein) was collected, concentrated, and loaded onto a Superdex 200 10/300 GL column equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.1 mM TCEP.
Unless otherwise stated, all proteins were concentrated, flash frozen in liquid nitrogen, and stored in aliquots at -80 °C.
Reconstitution of native CST-PP FL and MBP-POLA1 N -CST. Freshly purified PP FL and CST were mixed in equimolar amounts and incubated for 1 h at 4 °C prior to loading onto a HiLoad Superdex 200 16/600 PG column equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM TCEP, and 5% glycerol. The indicated fractions (Extended Data Fig. 1a) were pooled and concentrated. Purified CST was mixed with a twofold molar excess of His 6 -MBP-PreSc-POLA1 N and incubated for 1 h at 4 °C prior to being loaded onto a HiLoad Superdex 200 16/600 PG column equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM TCEP, and 5% glycerol. The indicated fractions (Extended Data Fig. 5) were pooled and concentrated.
Negative-stain EM sample preparation, data collection, and image processing. Protein samples for negative-stain EM (3.5-μL drops, in a concentration range of 0.01-0.05 mg/mL) were adsorbed to glow-discharged carbon-coated copper grids with a collodion film, washed with three drops of deionized water, and stained with two drops of freshly prepared 0.7% w/v uranyl formate. Samples were imaged at room temperature using a Phillips CM10 electron microscope equipped with a tungsten filament and operated at an acceleration voltage of 80 kV. The magnification used corresponds to a calibrated pixel size of 2.8 Å. Particle coordinates were auto-picked using the Swarm picker in EMAN2 (ref. 32  Cryo-EM sample preparation and data collection. Four microliters of the GraFix-stabilized samples were applied to Quantifoil R 1.2/1.3 mesh Au400 holey carbon grids covered with a graphene oxide support layer (EMS), blotted for 1-1.5 sec, and plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4 °C and 100% humidity.
Cryo-EM imaging was performed in the Cryo-EM Resource Center at the Rockefeller University using SerialEM 33 . Data-collection parameters are summarized in Table 1.
For CST-PP FL , one dataset (dataset 1) was collected on a 200-kV Talos Arctica electron microscope (Thermo Fisher Scientific) at a nominal magnification of ×28,000 (TEM nanoprobe), corresponding to a calibrated pixel size of 1.5 Å on the specimen level. Images were collected using a defocus range from −1.5 to −3 μm with a K2 Summit direct electron detector (Gatan) in super-resolution counting mode. Exposures of 10 sec were dose-fractionated into 50 frames (200 ms per frame) with a dose rate of 12 electrons/pixel/sec (approximately 1.07 electrons per Å 2 per frame), resulting in a total dose of 53 electrons per Å 2 . The second dataset (dataset 2) was collected on a 300-kV Titan Krios electron microscope at a nominal magnification of ×53,000 (EFTEM nanoprobe), corresponding to a calibrated pixel size of 1.32 Å on the specimen level. Images were collected using a defocus range from -1 to -2.2 μm with a K3 direct electron detector (Gatan) in super-resolution counting mode. Exposures of 3 sec were dose-fractionated into 50 frames (60 ms per frame) with a dose rate of 33 electrons/pixel/sec (approximately 1.14 electrons per Å 2 per frame), resulting in a total dose of 57 electrons per Å 2 .
For CST-PP ΔN , data were collected on a 300-kV Titan Krios electron microscope at a nominal magnification of ×64,000, corresponding to a calibrated pixel size of 1.08 Å on the specimen level. Images were collected using a defocus range from -1 to -2.2 μm with a K3 direct electron detector (Gatan) in super-resolution counting mode. Exposures of 3 sec were dose-fractionated into 50 frames (60 ms per frame), with a dose rate of 30 electrons/pixel/sec (approximately 1.03 electrons per Å 2 per frame), resulting in a total dose of 52 electrons per Å 2 .
Cryo-EM data processing. For all datasets, movie stacks were motion-corrected with the RELION-3 (ref. 34 ) implementation of MotionCor2 and motion-corrected micrographs were manually inspected and curated (graphene oxide coverage of grids were inconsistent) prior to CTF parameter estimation with CTFFIND4 (ref. 35 ) implemented in RELION-3. Particles were automatically picked with Gautomatch and extracted in RELION-3 for all further 2D and 3D processing steps. Auto-picked particles were examined by 2D classification, and particles in 'bad' classes corresponding to ice contamination or graphene oxide fold lines were discarded. The first reference model was generated using RELION-3's 3D initial model job and subsequently improved as continued 3D classification produced better maps.
For the CST-PP FL complex, multiple processing strategies were pursued to generate higher-resolution maps but were unsuccessful owing to substantial heterogeneity among the particles. The reported standard image-processing pipeline was performed with twofold binned images (to speed up computation), as the resolution of the resulting map did not approach the Nyquist limit. Because the two datasets were collected on different microscopes, RELION 3.1 was used to combine the particle stacks (imported as two separate optics groups). The combined particles were used for further 3D classification and 3D refinement (Extended Data Fig. 2).
For the CST-PP ΔN complex, a supervised 3D classification step in RELION-3 was introduced, using three references: CST-PP ΔN , PP ΔN alone, and a noise/ junk 'decoy' class. After discarding particles assigned to the latter two classes, the remaining particles were subjected to 3D classification into four classes using a single reference. Because particles in the class with the best-defined features still showed low CST occupancy, a consensus 3D refinement was performed and the resulting map segmented into CST and PP ΔN using UCSF Chimera 36 . Using a mask generated in RELION-3, the partial signal of PP ΔN was subtracted and the signal-subtracted particles were subjected to focused 3D classification without alignment. The final stack contained 131,850 particles showing intact CST. The particles were reverted to include PP ΔN , and used for three cycles of iterative 3D refinement, CTF refinement, and Bayesian polishing. The resulting density map was sharpened by post-processing, and Fourier shell correlation (FSC) curves and a local resolution map were calculated in RELION-3 (Extended Data Fig. 3).
Model building and refinement. An atomic model was built into the 4.6-Å resolution map of the CST-PP ΔN complex. The crystal structure of the apo PP ΔN (PDB ID: 5EXR) and cryo-EM structure of CST (PDB ID: 6W6W) were used for initial rigid-body docking into the map. The map showed only weak density corresponding to the C-terminal half of STN1 (STN1 C , 184-368 aa), likely owing to the ssDNA charge preventing STN1 C binding, as previously described 13 , and was therefore removed for model building. The geometry of PRIM1 in the PP ΔN crystal structure is poor and it was substituted in our initial model with a crystal structure of PRIM1 determined at higher resolution (PDB ID: 6RB4, alternate conformers removed 37 ). Although the geometries of the other PP subunits in the starting model (PDB ID: 5EXR) were also poor, we chose to continue with this model because it is the only experimentally determined model of apo POLA1/POLA2/PRIM2 in the occluded conformation and retains inter-subunit contact information that would not be captured by AlphaFold 2 models or other experimental structures of the subunits in isolation. The N-terminus of CTC1 was poorly resolved in the previously determined cryo-EM map and not modeled, so it was substituted with the AlphaFold 2 model of CTC1 (AF-Q2NKJ3-F1), which agrees with the experimentally determined structure of CTC1 (refs. 19,20 ). All subunits were docked into the cryo-EM map using the Chimera 'Fit in Map' tool, and the model was flexibly fitted using the ISOLDE plugin for ChimeraX 38,39 . After the model was improved by iterative cycles of refinement in Phenix (phenix.real_space_refine) and manual adjustment in Coot 40 , the geometry and map fit of the final model was validated (phenix.validation_cryoem) 41 .

Evolutionary conservation analysis.
All sequence accession numbers used in this study are listed in Supplementary Table 1. Protein sequences obtained from BLAST (blastp suite) were analyzed using Jalview 42,43 and multiple sequence comparison by log expectation (MUSCLE). Representative sequences for CTC1 and POLA1 were modeled in AlphaFold 2 (refs. 19,20 ) with the template database from 14 May 2020 and the casp14 preset. AlphaFold models were aligned in PyMOL (Schrödinger) and visualized in ChimeraX 39 .

Microscale thermophoresis.
Microscale thermophoresis experiments were performed on a NanoTemper Monolith NT.115 machine. All samples were buffer exchanged in centrifugal concentrators (Amicon Ultra-0.5 mL) into buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM DTT, and 0.05% (v/v) Tween-20. His 6 -MBP-CST was labeled according to manufacturer instructions with RED-tris-NTA second-generation dye (NanoTemper Technologies). Fifty nanomolar labeled His 6 -MBP-CST was incubated with serial dilutions of unlabeled POLA1 CTD constructs, and thermophoresis was measured at room temperature with an excitation power of 20% and an MST power of 20%. Titrations were performed in triplicate (experimental replicates), and capillary scans were performed in triplicate (technical replicates). Data were analyzed at the 10-sec time point with the MO Affinity Analysis Software (version 2.3, NanoTemper Technologies) using the K D fit option with outliers owing to aggregation automatically determined by the software. For CRL WT , the data were manually split to account for the presence of two binding events 23 . Reported K D values were calculated in the MO Affinity Analysis software and data were plotted with Prism 9 (GraphPad).
Mass spectrometry and data analysis. Samples were dialyzed against 100 mM ammonium bicarbonate, reduced with 50 mM TCEP at 60 °C for 10 min and alkylated with 50 mM iodoacetamide in the dark for 15 min at 37 °C. Digestion was carried out at 37 °C overnight with 125 ng/mL sequencing-grade modified trypsin (Fisher Scientific, PI90057) in 25 mM ammonium bicarbonate supplemented with ProteaseMax (Fisher Scientific, PRV2071). Reaction mix was supplemented with trifluoroacetic acid (TFA, Fisher Scientific, A116) to a final concentration of 0.1%. The resulting peptides were passed through C18 Spin Tips (Fisher Scientific, PI84850) before elution with 40 μL of 80% acetonitrile (ACN, Fisher Scientific, A9561) in 0.1% TFA. Eluted peptides were dried and resuspended in 20 μL 0.1% formic acid (FA; Fisher Scientific, A117) for MS analysis. Peptides were analyzed in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) coupled to an EASY-nLC (Thermo Scientific) liquid chromatography system, with a 2 μm, 500 mm EASY-Spray column. The peptides were eluted over a 120-min linear gradient from 96% buffer A (0.1 % FA in water) to 40% buffer B (0.1 % FA in ACN), then continued to 98% buffer B over 20 min with a flow rate of 250 nL/ min. Each full MS scan (R = 60,000) was followed by 20 data-dependent MS2 scans (R = 15,000) with high-energy collisional dissociation (HCD) and an isolation window of 2.0 m/z. Normalized collision energy was set to 35. Precursors of charge state ≤ 3 were collected for MS2 scans in enumerative mode, precursors of charge state 4-6 were collected for MS2 scans in cross-link discovery mode (both were performed for each sample); monoisotopic precursor selection was enabled and a dynamic exclusion window of 30.0 sec was set. Raw files obtained in enumerative mode were analyzed by pFind3 software 45 in open search mode and protein modifications inferred by pFind3 and comprising >0.5% of total protein were included as the variable modifications in pLink2 (ref. 46 ) search parameters. pLink2 results were filtered for FDR (<5%), e-value (<1 × 10 -3 ), score (<1 × 10 -2 ), and abundance (PSMs ≥ 5). Cross-links were visualized using xiNET 47 . Fig. 2 | Cryo-EM image-processing pipeline of CST-PP FL . a, Cryo-EM image-processing pipeline used for the CST-PP FL complex. Two datasets were combined in RELION 3.1 as separate optics groups, and particles were refined together with a reference generated from dataset no. 1. See Table 1 for full data collection and processing details. b, 2D-class averages from a cleanup step prior to 3D classification. Classes clearly representing ice contamination or graphene oxide edges were removed during cleanup. Fig. 3 | graFix preparation of CST-PP ΔN complex and analysis by cryo-EM. a, GraFix preparation of CST-PP ΔN -ssDNA. Top: Coomassie blue-stained SDS-PAGE gel (4-12% Tris-Glycine; Invitrogen) of GraFix fractions, showing formation of cross-linked CST-PP ΔN species. Fractions marked with an asterisk (*) were pooled for analysis by cryo-EM. Bottom: SYBR Gold-stained native PAGE gel (4-20% TBE; Invitrogen) of GraFix fractions, showing free and bound ssDNA. This reconstitution was independently reproduced at least three times with similar results. b, Selected high defocus motioncorrected cryo-EM micrograph of CST-PP ΔN (representative of 9,282 curated micrographs). c, Cryo-EM image-processing pipeline used for the CST-PP ΔN complex, including supervised 3D classification and focused 3D classification steps used to select particles with intact CST. Gold-standard FSC curves for the final map of CST-PP ΔN indicate a global resolution of 4.6 Å (FSC = 0.143). See Table 1 for full data collection and processing details. d, 2D-class averages obtained with the particle stack (131,850 particles) used to generate the final CST-PP ΔN map show blurring at the peripheral regions, indicating flexibility. e, Local resolution estimates of the CST-PP ΔN map. f, Orthogonal views of the CST-PP ΔN -ssDNA model fit into the cryo-EM map. Fig. 4 | analysis of the CTC1-PoLa1 CTD interface surfaces. Surface representations of the CTC1-POLA1 CTD interface shown in two orientations related by a 180° rotation and views CTC1 and POLA1 CTD after separation to reveal the interface surfaces (outlined in black). a, Surface overlaid with cartoon representation of CTC1-POLA1 CTD . Identification of CTC1 and POLA1 CTD binding surfaces, colored in dark blue. b, Surface representation colored according to electrostatic potential. c, Surface representation colored according to sequence conservation for metazoans using an alignment containing all species listed in Supplementary Table 1.