research papers
The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins
aDepartment of Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands, bLaboratory of Biology, School of Medicine, University of Patras, 26505 Rio, Patras, Greece, and cLaboratory of Physiology, School of Medicine, University of Patras, 26505 Rio, Patras, Greece
*Correspondence e-mail: a.perrakis@nki.nl
GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.
of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and the Geminin–Idas heterodimer structures. It is also shown that bothKeywords: Geminin; GemC1; Idas; McIdas; multicilin; coiled coil; DNA-replication license; protein stability.
PDB reference: GEMC1 coiled-coil domain, 5c9n
1. Introduction
Geminin coiled-coil domain-containing protein 1, GemC1, is a member of the Geminin superfamily. The three members of this family, Geminin, Idas and GemC1, all share a conserved coiled-coil domain.
Geminin was the first to be identified, as an inhibitor of DNA replication (McGarry & Kirschner, 1998; reviewed in Caillat & Perrakis, 2012). The binding of Geminin to Cdt1 inhibits the loading of the mini-chromosome maintenance complex (MCM) onto chromatin and pre-replication complex (preRC) formation (Tada et al., 2001; Wohlschlegel et al., 2000; reviewed in Lygerou & Nurse, 2000; Symeonidou et al., 2013). Besides its role in proliferation, Geminin also has a role in cell differentiation (Seo & Kroll, 2006; Champeris Tsaniras et al., 2014). The coiled coil of Geminin resides in the middle of the protein and assembles in a head-to-head coiled-coil homodimer that binds one molecule of Cdt1 (De Marco et al., 2009; Lee et al., 2004; Saxena et al., 2004).
Idas (also referred to as multicilin and McIdas) was identified as a protein that interacts with Geminin, exhibits high levels of expression in the mouse forebrain and regulates DNA replication and centrosome numbers (Pefani et al., 2011). Idas has also been identified as a key regulator of multiciliate cell differentiation that drives centriole biogenesis (Ma et al., 2014; Stubbs et al., 2012). Idas preferentially interacts with Geminin than with itself, forming a tight heterodimer between the two coiled-coil domains (Caillat et al., 2013).
GemC1 has been identified as a Geminin homologue, and is also implicated in DNA replication but at a later stage than Geminin. GemC1 has been shown to mediate TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins, enabling pre-initiation complex formation and initiation of DNA replication (Balestrini et al., 2010).
The mechanism by which Geminin is able to coordinate both cell proliferation and cell differentiation is not fully understood (Caillat & Perrakis, 2012; Champeris Tsaniras et al., 2014). Having previously shown that Idas can preferentially interact with Geminin through its coiled-coil domain and that this interaction is important for Idas function (Caillat et al., 2013; Pefani et al., 2011), we sought to examine the structure of GemC1 and how this might explain its function and the relationships within the Geminin family.
2. Materials and methods
2.1. Cloning, expression and purification
A synthetic gene (GenScript) harbouring a codon-optimized DNA sequence (according to the manufacturer's protocols) was used for all human GemC1 (UniProt ID A6NCL1) constructs. The constructs for GemC1, tGemC1 (64–146), dGemC1 (29–240), GemC1_C-ter (241–334) and full-length GemC1 (1–334), were cloned into the pETNKI-His-3CLIC-kan vector (Luna-Vargas et al., 2011) for expression with a cleavable His tag. The constructs for Geminin, full-length Geminin, dGeminin (29–209) and tGeminin (82–160), and the Idas construct used for the purification of tIdas–tGeminin and tIdas–tIdas dimers have been described previously (Caillat et al., 2013; De Marco et al., 2009). To express the tGemC1–tGeminin heterodimer, we used the tGemC1 construct described above together with a tGeminin construct that we have described previously (Caillat et al., 2013) and inserted it into the pET-22b (Novagen) vector for expression without a tag. As these two plasmids are resistant to kanamycin and ampicillin, respectively, they allow efficient co-expression experiments. The two mutations in GemC1, L123E and L130E (tGemC1L123,130E), were generated using the QuikChange site-directed mutagenesis kit (Stratagene). All complexes were purified by IMAC and in a buffer consisting of 50 mM HEPES–NaOH pH 7.5, 150 mM NaCl, 0.5 mM TCEP; detailed protocols are available in Caillat et al. (2013) and De Marco et al. (2009). We note that the heterodimers are purified with a tag on either GemC1 or Idas; as untagged Geminin is the more abundantly expressed protein in our experiments, purification of the less abundant protein practically ensures purification of the heterodimer. All proteins were further purified by and the final product was examined by Coomassie Brilliant Blue-stained polyacrylamide gel to confirm that an approximately stoichiometric amount of complex was the final purification product.
2.2. Multi-angle laser light scattering
Multi-angle laser −1 were injected onto the column. Data analysis was carried out with ASTRA using a dn/dc value of 0.185. runs for tGemC1–tGeminin were performed in a Superdex 75 HR 10/30 column attached to an ÄKTApurifier.
(MALLS) experiments were performed in a Superdex 75 HR 10/30 column attached to an ÄKTA FPLC and coupled to a miniDAWN light-scattering detector (Wyatt Technology) and a Dn-1000 differential refractive-index detector (WGE Dr Bures). 100 µl of purified tGemC1 dimer at a concentration of ∼2.0 mg ml2.3. Mammalian cell culture, transfection and immunoprecipitation
HA-tagged GemC1, Geminin-GFP, Geminin(1–72)-GFP, Idas-GFP and Cdt1-GFP were cloned in pcDNA3.1 for expression in mammalian cells. U2OS cells were cultured in DMEM (Invitrogen) with 10% foetal bovine serum (Invitrogen). Cells were transfected with the TurboFect transfection reagent (Fermentas) according to the manufacturer's instructions. U2OS cells were transfected with GEMC1-HA and other constructs as indicated and were collected 24 h post-transfection. Immunoprecipitation of GEMC1-HA was performed using an anti-HA antibody (12CA5, Santa Cruz) as described in Pefani et al. (2011). Immunoprecipitates and total cell extracts corresponding to 10% of immunoprecipitates were analysed by Western blotting using anti-HA (Molecular Probes), anti-GFP and anti-Geminin (Xouri et al., 2004; Iliou et al., 2013) antibodies.
2.4. Tm determination based on tryptophan fluorescence (OPTIM 1000)
Thermal unfolding and aggregation curves were measured in 25 mM HEPES–NaOH pH 7.5, 150 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine at a concentration of 1 mg ml−1 using an OPTIM 1000 from Avacta.
The barycentric mean fluorescence was calculated according to
where λbcm is the barycentric mean, λ is the wavelength, Fλ is the fluorescence intensity at wavelength λ, m = 300 nm and n = 450 nm.
The static light-scattering signal was also recorded from the samples to detect the presence of aggregates.
2.5. Analysis of the stability of the coiled coil by (CD)
Far-UV CD experiments were performed on a J-810 spectropolarimeter (Jasco) with a Peltier thermocontrol element (Jasco). CD data were recorded at a fixed wavelength of 220 nm with a linear temperature gradient from 10 to 90°C. All samples were adjusted to a concentration of ∼0.3 mg ml−1. No visual precipitation was observed after completion of the experiment. Data analysis was performed using the formulae described in Greenfield (2006) as implemented in GraphPad Prism by the authors.
2.6. Crystallization
Screening was performed using previously described procedures (Newman et al., 2005) in 96-well sitting-drop vapour-diffusion plates (MRC 2-Well Crystallization Plate manufactured by Swissci). Following optimization, crystals used for diffraction studies were grown at 4°C, mixing 200 nl 10 mg ml−1 tGemC1L123,130E with 200 nl 0.1 M HEPES buffer pH 7.5, 7% ethanol, 10% 2-methyl-2,4-pentanediol (MPD), 0.01 M ethylenediaminetetraacetic acid disodium salt dihydrate. Crystals were soaked in the reservoir solution supplemented with MPD to a final concentration of 32%(w/v) and were vitrified by plunging into liquid nitrogen.
2.7. Data collection, structure solution and refinement
Diffraction data were collected on beamline ID23-2 at the ESRF at a wavelength of 0.8726 Å. Intensity integration and scaling was performed using the XDS package (Kabsch, 2010). The structure was solved by with Phaser (McCoy, 2007) using a polyalanine model of dimeric Geminin (PDB entry 2wvr ; De Marco et al., 2009) as the search model. One homodimer of tGemC1L123,130E was present in each of the P212121 The model was rebuilt in the map resulting from the molecular-replacement solution using ARP/wARP (Langer et al., 2008) and manually adjusted in Coot (Emsley et al., 2010). was performed using phenix.refine (Afonine et al., 2012) and in later stages using the PDB_REDO web server (Joosten et al., 2014) incorporating REFMAC (Murshudov et al., 2011). Statistics of data reduction and structure are presented in Table 1.
|
3. Results and discussion
3.1. The structure of the GemC1 coiled coil
Expression trials of full-length GemC1 (1–334) and an extended construct encompassing a `long' predicted coiled-coil domain (29–208; dGemC1) resulted in insoluble protein. Expression of a construct slightly longer than the predicted coiled-coil domain (64–146; tGemC1) resulted in protein that was soluble at concentrations below 1 µM. Several tags (GST, SUMO and Trigger Factor) did not improve the solubility. Examining the helical wheel prediction diagram of the GemC1 coiled-coil homodimer, we observed that some hydrophobic residues are not in the core interface (register positions a and d), but are instead exposed to the solvent. In particular, at the C-terminal end of the coiled coil, residues Leu119, Val120, Leu123, Ala127, Leu130 and Leu131 constitute a hydrophobic patch. We hypothesized that this hydrophobic patch could lead to aggregation of the GemC1 protein. We thus decided to mutate residues Leu123 and Leu130 to glutamates to increase the solubility. It should be noted that some predictions place these residues in the d position of the coiled coil; however, these predictions assume a coiled-coil irregularity in the 113–114 region, something that is unlikely based on the structures of the homologous coiled coils of Geminin and Idas. Our mutations yielded the construct tGemC1L123,130E, which allowed the expression of highly soluble protein (>2 mM). The protein behaved as a dimer in a coupled to multi-angle laser (MALLS) experiment (Fig. 1a).
tGemC1L123,130E (from here on we will refer to this construct as tGemC1 for simplicity) was crystallized and the structure was determined to 2.2 Å resolution and refined to an Rfree of 24.9% with excellent geometry (Table 1). The structure showed a typical dimeric parallel coiled-coil homodimer (Figs. 1b and 1c), with two α-helices that pack together in a left-handed superhelix. Both chains have about 20 disordered residues in the C-terminus and five disordered residues in the N-terminus, as only residues 69–132 and 69–129 are well resolved in the electron density in each of the two chains. Residues 71–129 and 70–124 are in α-helical conformation in each chain. The two mutated leucine residues are indeed pointing to the solvent, as expected from our sequence analysis and in contrast to the other predictions discussed above. Although we cannot formally exclude that our mutations changed the coiled coil, this is very unlikely as we observe regular helices and the coiled coil stops in approximately the same place as in the homologous structures of the Geminin (PDB entry 1uii ; Thépaut et al., 2004) and Geminin–Idas (PDB entry 4bry ; Caillat et al., 2013) coiled coils. Based on our structural data, we conclude that the change of the hydrophobic solvent-exposed Leu123 and Leu130 to hydrophilic glutamate residues improved solubility without affecting the global structure.
Analysis of the structure using the SOCKET software (Walshaw & Woolfson, 2001) shows that the coiled-coil region extends from residues 73 to 115 and spans six heptads (technically speaking, one residue of a seventh heptad is present). In position d4, Lys97 does not form a `knobs-into-holes' interaction, forming a minor but characteristic irregularity in the series of interactions in the length of the coiled coil (Fig. 1c).
The structure of the Geminin coiled-coil homodimer as well as the structure of the Geminin–Idas coiled-coil heterodimer have previously been determined (Thépaut et al., 2004; Caillat et al., 2013). Comparing these structures with that of the tGemC1 homodimer (Fig. 2) shows several interesting features. Firstly, all three structures are composed of coiled coils of similar length, with Geminin having a more extended coiled coil (six full heptads with a four-residue N-terminal extension and a one-residue C-terminal extension) and Idas–Geminin a less extended coiled coil (five core heptads with two N- and C-terminal flanking regions of four residues each); GemC1 is intermediate in length. Analysis of the coiled-coil parameters by the program CCCP (Grigoryan & Degrado, 2011) shows that the GemC1 coiled coil has an ω0 angle of −4.1° per residue, suggesting a relatively tight left-handed superhelix compared with the Geminin homodimer (ω0 = −3.9° per residue) and Idas–Geminin (ω0 = −3.7° per residue). The superhelical radius (the distance from the superhelix axis to the helical axis of the chains) is longer in GemC1 at 5.1 Å compared with 4.7 Å for both Geminin and Idas–Geminin. The surface buried at the interface of GemC1 (1370 Å2) is slightly less than for Idas–Geminin (1463 Å2) and Geminin–Geminin (1572 Å2).
Similarly to both Geminin and Geminin–Idas, GemC1 has several nonhydrophobic residues in the a and d register positions: d1, d2, a1, d4 and a6. In addition, GemC1 has a highly unusual cysteine residue at position a1 (an alanine in both Geminin and Idas). The residue in position d1 is the negatively charged Glu76 in GemC1 and is followed by Glu77; this is sharply opposed to the positively charged pair of Arg106 and Arg107 residues in Geminin and the Asn189 and Gln190 polar pair in Idas (Fig. 3a). The Glu76 in GemC1 creates an electrostatic repulsion with Glu77 from the second monomer in GemC1, resulting in the two helices of the coil being further apart than in the other structures. In position d2, GemC1 has a Gln in place of an Ala in the other two structures. This Gln83 is involved in a nonsymmetric network of side-chain interactions that also involves the well conserved Asn87 in position a1. The Lys97 residue in position d4, which is fully conserved in Geminin and Idas, interacts with Glu98 in position e4 of the opposing chain; in the Geminin and Idas structures this is Asp128 (Fig. 3b). Apparently, maintaining the hydrogen-bonding interaction with the longer Glu98 places Lys97 in GemC1 in a more extended conformation that is incompatible with the definition of the `knobs-into-holes' geometry for coiled coils (see above), but still maintains hydrogen-bonding interactions between the monomers, suggesting that this residue is not crucial for coiled-coil formation and that the lack of the `knobs-into-holes' structure is rather an anomaly and not a defining feature of the GemC1 coiled coil. Finally, the Asn108 in position a6 is conserved in the family and is involved in a stabilizing hydrogen bond between the two chains.
3.2. GemC1 and Geminin interact through the coiled-coil domain
We have previously shown that Idas prefers to interact with Geminin and form a heterodimer than to homodimerize (Caillat et al., 2013; Pefani et al., 2011). To determine whether the same holds true for GemC1, we first co-expressed His-tagged GemC1 and Geminin and were able to purify a stoichiometric complex between the two proteins (Fig. 4a). This is also notable because GemC1 alone was never soluble in our expression trials. In addition, the coiled coil of Geminin (tGeminin) was sufficient to solubilize the coiled-coil domain of GemC1 (tGemC1) and of the longer dGemC1, but was not sufficient to solubilize full-length GemC1. These results indicate that GemC1 and Geminin interact through their coiled-coil domains but are likely to have more extended interactions, as full-length GemC1 needs full-length Geminin to stabilize. Notably, even when GemC1 is more abundant than Geminin in the cell lysates purification through the His tag attached to GemC1 results in an approximately stoichiometric 1:1 complex between GemC1 and Geminin (Fig. 4a), suggesting that at least under these specific conditions the GemC1–Geminin complex is preferred. Finally, expression and purification of the tGemC1–tGeminin complex (by IMAC on the His tag on GemC1 alone) resulted in a complex that subsequently ran as a single peak on a column (Fig. 4b), with a retention volume directly comparable to that of the tGemC1–tGemC1 homodimer (Fig. 1a), suggesting that tGemC1 and tGeminin fold as a stable stoichiometric heterodimer.
To test whether GemC1 also interacts with Geminin in human cells, we transfected U2OS cells with a construct expressing GemC1-HA. The transfected GemC1-HA is able to co-precipitate the endogenous Geminin (Fig. 4c), indicating that GemC1 and Geminin also interact in human cells. To further determine whether this interaction is dependent on the coiled-coil domain of Geminin, we made a Geminin(1–72) construct encompassing the N-terminal 72 amino acids of Geminin and lacking the coiled-coil domain, and transfected Geminin and Geminin(1–72) as GFP fusions together with GemC1-HA in U2OS cells. The transfected GemC1-HA is able to co-precipitate GFP-Geminin but not GFP-Geminin(1–72) (Fig. 4d), indicating that the Geminin coiled coil is necessary for the interaction.
We then wanted to check whether GemC1 also interacts with Idas. For this, we used co-transfection of U2OS cells with GemC1-HA and GFP-tagged Idas. A weak interaction between GemC1 and Idas was observed under these conditions (Fig. 5), in which GemC1-HA was only able to co-precipitate a small fraction of the total Idas-GFP protein. However, we were unable to produce any GemC1–Idas complex from bacteria for in vitro studies. In a parallel experiment, we also checked whether GemC1 binds Cdt1, the major partner of Geminin, but we were unable to observe an interaction.
Next, we wanted to study the stability of the GemC1 homodimers and heterodimers in comparison with other dimers formed by the Geminin-like family of proteins.
3.3. On the stability of the Geminin-family coiled coils
We have collectively shown that the three family members, Geminin, Idas and GemC1, can form homodimers and that Idas and GemC1 can form heterodimers with Geminin through their coiled-coil domains. Although we were able to observe a weak Idas–GemC1 interaction in human cells, we were unable to produce any form of such a recombinant complex in order to check its stability. We have previously studied the stability of the Idas and Geminin coiled-coil dimers and concluded that the tIdas–tIdas dimer was unstable under physiological conditions, while tGeminin–tGeminin and tIdas–tGeminin were stable proteins (Caillat et al., 2013).
We first checked the stabilities of all five dimers (tGeminin–tGeminin, tIdas–tIdas, tGemC1–tGemC1, tIdas–tGeminin and tGemC1–Geminin) using the OPTIM 1000 instrument, monitoring tryptophan fluorescence to estimate the stability of the dimers. While we were able to accurately reproduce our previous results (Table 2), unanticipated curves were obtained for the tGemC1-containing complexes (Fig. 6a). Structural information can provide a biophysical explanation for this unexpected behaviour: the hydrophobic surfaces of the Trp99 and Trp182 residues in Geminin and Idas are very well buried between neighbouring side chains of the coiled coil (Fig. 6b), but in GemC1 Trp75 is surface-exposed. It is important to note that while this tryptophan is unique in the N-terminus of all three coiled-coil sequences, it is not actually conserved and is not in the same heptad nor in the same coil register (d in Geminin and Idas and c in GemC1). Thus, the environment of Trp75 in GemC1 will hardly change upon unfolding and no signal should be visible in the melting curves. Careful analysis of the melting curves supports this theory: while for the tGemC1 homodimer the signal decreases steadily without a clear deflection point, for the tGemC1–tGeminin complex there is a signal increase at about 65°C, similar to that owing to the unfolding of the tGeminin homodimer, which is likely to come from the complete melting of the Geminin chain at this temperature.
|
To examine the stability of GemC1 complexes without using the tryptophan-fluorescence signal, we resorted to the well established method of c) that while the three homodimers unfold in a single state, the two heterodimers unfold in two states, with each helix presumably having a different melting point. Analyzing the data, we assumed that the coiled coils unfold as a dimer (Greenfield, 2006), as they are not interlinked by covalent bonds, in a single step or in two steps, depending on their homodimeric or heterodimeric state. This analysis clearly shows that tGemC1, with a Tm of 34.6°C, is not as stable as tGeminin (65.3°C) but is significantly more stable than Idas (26.9°C). Still, this value suggests that the tGemC1 homodimer should be rather unstable in physiological environments; as we have not been able to obtain soluble full-length tGemC1 to perform this experiment, we cannot be confident whether this conclusion can be extended to the wild-type protein. However, our data suggest that GemC1 alone may be unstable and may be unlikely to be present in cells as a homodimer on its own under physiological conditions. We speculate that GemC1 may exist as a heterodimer with Geminin in cells, while complex formation with other partners, or post-translational modification, may be required to stabilize a GemC1 homodimer. The tGemC1–tGeminin complex shows a two-state unfolding: the first event is at 42.6°C and the second event at 62.6°C. As we obtain an excellent fit presuming that that the ratio between the two events is equal to the ratio of total change in the ellipticity of unfolding between tGemC1 and tGeminin, we interpret the first event as the unfolding of tGemC1 (which has been stabilized by about 8°C owing to interaction with Geminin) and the second event as the unfolding of tGeminin, which has been moderately destabilized. This result implies that when GemC1 and Geminin are co-expressed in cells the predominant form of GemC1 is likely to be in complex with Geminin, as previously suggested for Idas. Interestingly, the CD data also show a two-event curve for the tIdas–tGeminin complex. However, in this case the associated molar ellipticity change for the first event is very small and we think that this is likely to be an unfolding of the tIdas C-terminus that is not part of the coiled coil; the two helices in the tIdas–tGeminin complex unfold at similar temperatures, but the data cannot be deconvoluted. The same could hold true for the tGemC1–tGeminin unfolding but to a much lesser degree, as there we more clearly see the two states which are more likely to correspond to the two helices.
(CD). As the coiled coil is helical, we chose to study the of the helices, monitoring the ellipticity at 222 nm. It was evident from the melting curves (Fig. 6Some interesting hypotheses could be extracted by comparing the OPTIM and CD experiments: owing to the positioning of the tryptophan residue OPTIM monitors the unfolding of the N-terminal region of the coiled coils, while the CD gives a more global picture. For tGeminin and tIdas, it is clear that the Tm obtained from OPTIM (corresponding to the N-terminal part) is higher than that obtained from CD (corresponding to the complete coiled coil): this could imply that these coiled coils unfold from the C-terminus towards the N-terminus. Presuming that the folding takes the same pathway as the unfolding that we study here, this would in turn imply that these coils also fold from the N-terminus towards the C-terminus, favouring our previous hypothesis of co-translational assembly of the heterodimeric Idas–Geminin complex (Caillat et al., 2013) and leading us to propose that the same holds true for the GemC1–Geminin complex. The biophysical issues around coiled-coil folding are considerable (for a review, see Lupas & Gruber, 2005) and sophisticated approaches have been used to study these problems. Thus, the above conjecture should be taken with caution. However, we believe that monitoring the unfolding through the two different signals that we use in this case (for the first time, to our knowledge) provides novel insight into how the Geminin-family coiled coils might fold.
In conclusion, our structure of the GemC1 coiled coil, together with biophysical data, suggest that the GemC1 coiled coil is likely to be unstable and (as for Idas) a GemC1–Geminin dimer might be a more stable structure in cells. Our results thus reinforce the concept that both Idas and GemC1 may modulate the abundance of the Geminin dimer when co-expressed in cells. The GemC1–Geminin and Idas–Geminin heterodimers are likely to be major pools of Idas and GemC1 in cells which co-express Geminin, such as proliferating cells, and could modulate the diverse functions of Geminin, Idas and GemC1 in proliferation and differentiation.
Acknowledgements
We would like to thank Julian Blow for interesting discussions about GemC1, Jonas M. Dörr for help with the CD experiments and Eleonore von Castelmur and Tatjana Heidebrecht for help with crystal and protein production. Diffraction data were collected on ID23-2 at the ESRF. Work in the laboratory of ZL was supported by the European Research Council (DYNACOM, 281851).
References
Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Balestrini, A. A., Cosentino, C. C., Errico, A. A., Garner, E. E. & Costanzo, V. V. (2010). Nature Cell Biol. 12, 484–491. CrossRef CAS PubMed Google Scholar
Caillat, C., Pefani, D.-E., Gillespie, P. J., Taraviras, S., Blow, J. J., Lygerou, Z. & Perrakis, A. (2013). J. Biol. Chem. 288, 31624–31634. CrossRef CAS PubMed Google Scholar
Caillat, C. & Perrakis, A. (2012). Subcell. Biochem. 62, 71–87. CrossRef CAS PubMed Google Scholar
Champeris Tsaniras, S., Kanellakis, N., Symeonidou, I. E., Nikolopoulou, P., Lygerou, Z. & Taraviras, S. (2014). Semin. Cell Dev. Biol. 30, 174–180. CrossRef CAS PubMed Google Scholar
De Marco, V. et al. (2009). Proc. Natl Acad. Sci. USA, 106, 19807–19812. CrossRef PubMed CAS Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. Web of Science CrossRef CAS IUCr Journals Google Scholar
Greenfield, N. J. (2006). Nature Protoc. 1, 2527–2535. Web of Science CrossRef CAS Google Scholar
Grigoryan, G. & Degrado, W. F. (2011). J. Mol. Biol. 405, 1079–1100. CrossRef CAS PubMed Google Scholar
Iliou, M. S., Kotantaki, P., Karamitros, D., Spella, M., Taraviras, S. & Lygerou, Z. (2013). Mech. Ageing Dev. 134, 10–23. CrossRef CAS PubMed Google Scholar
Joosten, R. P., Long, F., Murshudov, G. N. & Perrakis, A. (2014). IUCrJ, 1, 213–220. CrossRef CAS PubMed IUCr Journals Google Scholar
Kabsch, W. (2010). Acta Cryst. D66, 125–132. Web of Science CrossRef CAS IUCr Journals Google Scholar
Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. (2008). Nature Protoc. 3, 1171–1179. Web of Science CrossRef CAS Google Scholar
Lee, C., Hong, B., Choi, J., Kim, Y., Watanabe, S., Ishimi, Y., Enomoto, T., Tada, S., Kim, Y. & Cho, Y. (2004). Nature (London), 430, 913–917. CrossRef PubMed CAS Google Scholar
Luna-Vargas, M. P. A., Christodoulou, E., Alfieri, A., van Dijk, W. J., Stadnik, M., Hibbert, R. G., Sahtoe, D. D., Clerici, M., Marco, V. D., Littler, D., Celie, P. H. n., Sixma, T. K. & Perrakis, A. (2011). J. Struct. Biol. 175, 113–119. CAS PubMed Google Scholar
Lupas, A. N. & Gruber, M. (2005). Adv. Protein Chem. 70, 37–78. CrossRef PubMed CAS Google Scholar
Lygerou, Z. & Nurse, P. (2000). Science, 290, 2271–2273. PubMed CAS Google Scholar
Ma, L., Quigley, I., Omran, H. & Kintner, C. (2014). Genes Dev. 28, 1461–1471. CrossRef CAS PubMed Google Scholar
McCoy, A. J. (2007). Acta Cryst. D63, 32–41. Web of Science CrossRef CAS IUCr Journals Google Scholar
McGarry, T. J. & Kirschner, M. W. (1998). Cell, 93, 1043–1053. CrossRef CAS PubMed Google Scholar
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Newman, J., Egan, D., Walter, T. S., Meged, R., Berry, I., Ben Jelloul, M., Sussman, J. L., Stuart, D. I. & Perrakis, A. (2005). Acta Cryst. D61, 1426–1431. Web of Science CrossRef CAS IUCr Journals Google Scholar
Pefani, D.-E., Dimaki, M., Spella, M., Karantzelis, N., Mitsiki, E., Kyrousi, C., Symeonidou, I. E., Perrakis, A., Taraviras, S. & Lygerou, Z. (2011). J. Biol. Chem. 286, 23234–23246. CrossRef CAS PubMed Google Scholar
Saxena, S., Yuan, P., Dhar, S. K., Senga, T., Takeda, D., Robinson, H., Kornbluth, S., Swaminathan, K. & Dutta, A. (2004). Mol. Cell, 15, 245–258. CrossRef PubMed CAS Google Scholar
Seo, S. & Kroll, K. L. (2006). Cell Cycle, 5, 374–379. CrossRef PubMed CAS Google Scholar
Stubbs, J. L., Vladar, E. K., Axelrod, J. D. & Kintner, C. (2012). Nature Cell Biol. 14, 140–147. CrossRef CAS PubMed Google Scholar
Symeonidou, I. E., Kotsantis, P., Roukos, V., Rapsomaniki, M. A., Grecco, H. E., Bastiaens, P., Taraviras, S. & Lygerou, Z. (2013). J. Biol. Chem. 288, 35852–35867. CrossRef CAS PubMed Google Scholar
Tada, S., Li, A., Maiorano, D., Méchali, M. & Blow, J. J. (2001). Nature Cell Biol. 3, 107–113. CrossRef PubMed CAS Google Scholar
Thépaut, M., Maiorano, D., Guichou, J.-F., Augé, M.-T., Dumas, C., Méchali, M. & Padilla, A. (2004). J. Mol. Biol. 342, 275–287. Web of Science PubMed Google Scholar
Walshaw, J. & Woolfson, D. N. (2001). J. Mol. Biol. 307, 1427–1450. Web of Science CrossRef PubMed CAS Google Scholar
Wohlschlegel, J. A., Dwyer, B. T., Dhar, S. K., Cvetic, C., Walter, J. C. & Dutta, A. (2000). Science, 290, 2309–2312. CrossRef PubMed CAS Google Scholar
Xouri, G., Lygerou, Z., Nishitani, H., Pachnis, V., Nurse, P. & Taraviras, S. (2004). Eur. J. Biochem. 271, 3368–3378. CrossRef PubMed CAS Google Scholar
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.