Optimizing Protein Complexes for Crystal Growth† Bing Xiao,*,‡ C. Tarricone,§ Kuang Lin,| Geoff Kelly,‡,| and Neil Justin‡ DiVision of Molecular Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom, Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy, and Biomathematics & Statistics Scotland, JCMB, The UniVersity of Edinburgh, Edinburgh EH9 3JZ, Scotland, United Kingdom
CRYSTAL GROWTH & DESIGN 2007 VOL. 7, NO. 11 2213–2218
ReceiVed July 26, 2007; ReVised Manuscript ReceiVed September 19, 2007
ABSTRACT: Many intracellular proteins do not work on their own but rather in complex with small molecules, DNA, or other proteins. To gain a more fundamental understanding of protein interactions and their resulting functions, one requires a detailed structural model of relevant complexes. The first step in this challenge is to grow well-diffracting crystals. Three examples of protein complex crystallization will be discussed in detail below. In the first example, biophysical techniques such as fluorescence titration, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS) are used to characterize the protein and assess the most suitable conditions for complex formation. The second example utilizes bioinformatic information and proteomic techniques to engineer constructs of the protein that are most favorable for crystallization. The final example uses NMR information for optimizing complex-forming conditions, which allowed the growth of better-diffracting complex crystals. Introduction The formation of complexes by proteins is essential for normal cellular function. To gain a molecular understanding of protein interactions, it is required that crystals of proteins in complex are grown for X-ray structure determination. In recent years, the topics of preparation, characterization, crystallization, and structural analysis of complexes have been discussed and reviewed from different perspectives. Some examples of particular relevance from the recent crystallographic meetings have been described in detail.1–7 Here, we are focusing on our three experiments of how protein complexes can be optimized for crystallization in detail. The first example is illustrated by the crystallization of Arfaptin in complex with Rac1. Biophysical techniques such as isothermal titration calorimetry (ITC) and fluorescence titration are used to determine the most suitable conditions for complex formation. In addition, the technique of dynamic light scattering (DLS) was used to characterize the complex homogeneity and estimate its stability over the period of time required for crystallization. The complex of retinoblastoma tumor suppressor (Rb) with a peptide from the transcription factor E2F1 is used as the second example; here, bioinformatics information and proteomic techniques were used to engineer constructs of the protein that were more favorable for crystallization. In the third example, the SET domain protein prSET7 is complexed with various histone peptides; NMR information was then used for optimizing the complex-forming conditions. This allowed a stable ternary complex to be produced, which resulted in crystals that diffracted to a higher resolution. 1. Using Biophysical Techniques To Determine the Most Suitable Conditions for Complex Formation Arfaptin is a signaling protein, with a role mediating crosstalk between members of the Rho and Arf small GTPase † Part of the special issue (vol 7, issue 11) on the 11th International Conference on the Crystallization of Biological Macromolecules, Que´bec, Canada, August 16–21, 2006 (preconference August 13–16, 2006). * Corresponding author. Telephone: 442088162521. Fax: 442088162580. E-mail:
[email protected]. ‡ National Institute for Medical Research. § European Institute of Oncology. | The University of Edinburgh.
Figure 1. Structure of Arfaptin shown in ribbons representation. One monomer of the Arfaptin dimer is coloured blue and the other in yellow.
families. It has been shown to bind to Rac, Arf1, and Arf6 by different groups.8,9 To understand its role in these different signaling pathways, we undertook a structural and biochemical study on Arfaptin looking at the complex with Rac1. Crystals of Arfaptin (residues 118–341) were readily grown from solutions of a screening kit (Molecular Dimensions Ltd.). Crystals were obtained using the vapor diffusion technique in hanging drops using a 1:1 mixture of protein (∼10 mg/mL) and reservoir solution containing 0.1 M trisodium citrate dihydrate, 10% (v/v) isopropanol and 50 mM HEPES pH 7.5 at 4 °C. The structure was solved by MAD phasing10 and revealed that the molecule of Arfaptin exists as a banana-shaped dimer with an end to end distance of about 146 Å. Each of the monomers consists of three alpha-helices in a coiled-coil fold, and the two monomers are arranged as an extended antiparallel R-helical bundle (Figure 1). The next step was to crystallize Arfaptin in complex with Rac1. Thus, determining the binding affinity and stoichiometry of the Arfaptin–Rac1 complex was necessary for preparation of the complex for crystallization. Isothermal titration calorimetry (ITC) was used for this purpose. ITC is a thermodynamic technique for monitoring the interactions between two molecules; measurement of the heat generated or absorbed during a reaction allows accurate determination of binding constants (KB), reaction stoichiometry (n), enthalpy (∆H), and entropy (∆S) in a single experiment. For this experiment, the titrations were performed at 22 °C and Rac1 (at 1.0 mM) was titrated into 0.12 mM Arfaptin. The resulting data shows that Arfaptin
10.1021/cg7007039 CCC: $37.00 2007 American Chemical Society Published on Web 10/26/2007
2214 Crystal Growth & Design, Vol. 7, No. 11, 2007
Reviews Table 1. Results of Fluorescence Titration Experiments Showing Binding of Arfaptin to Rac1 at Various pH Conditionsa pH
Kd
6.0 7.0 8.0 9.0
10.22 7.51 5.94 3.36
a Buffer: 100 mM phosphate with 150 mM NaCl. All measurements were performed using an ISS spectrophotometer.
Figure 3. Structure of Rac–Arfaptin complex. The Rac–Arfaptin complex is shown in ribbon representation, with Arfaptin in the same orientation and color as shown in Figure 1. The Rac molecule is shown in green with the nucleotide in stick representation.
Figure 2. Arfaptin binding studies. Isothermal titration calorimetry measurements of Rac1 binding to Arfaptin. Top, raw data of ITC experiments performed at 22 °C. Bottom, integrated heat changes, corrected for the heat of dilution, and the fitted curve based on a singlesite model. (A) Data for the titration of Arfaptin into Rac.GMPPNP, which gives a Kd of 4.5 µM and a binding ration of 2. (B) Data for the titration of Rac.GDP into Arfaptin, which gives a Kd of 3 µM and a binding ration of 0.4.
binds to RacGMPPNP and RacGDP with dissociation constants (Kd) of 4.5 and 3 µM, respectively, and a stoichiometry of one G protein per Arfaptin dimer (Figure 2). Therefore, on the basis of these results, the crystallization complex to be used for screening was made by mixing equal volumes of Arfaptin (at 1 mM) and Rac1 (at 0.5 mM). Altogether, 27 different types of
crystals were produced from various screening kits (including Crystal Screen I and II, Natrix (Hampton Research); Wizard I and II (Emerald BioStructure Inc.)) at two temperatures (4 and 18 °C). The contents of the crystals were determined using SDSPAGE or by X-ray analysis. However, none of these crystals produced contained the complex, but rather Rac1 or Arfaptin on their own. To maximize the chance of obtaining complex crystals, fluorescence anisotropy (FA) titration experiments were then used to find better buffer conditions for the complex preparation. FA allows the use of much smaller amounts of proteins than ITC. The titration results are shown in the Table 1. The complex has a slightly tighter binding affinity at higher pH, and hence, it is not surprising that the individual components readily crystallized from the screening kits at pH 5.0–7.5. Thus, the new strategy was to crystallize the complex using buffers that have a pH in the region of 8.0–10.0. Solubility tests of the complexes in different crystallization conditions revealed that polyethylene glycols (PEG) are more favorable than salts as precipitants for the complex, so PEGs at various molecular weights were selected for the crystallization. Crystals of the complex grew over 1 month using 0.5 µL of protein solution mixed with 0.5 µL of reservoir buffer containing 0.1 M Tris pH 9.0 or 9.5 and 10% PEG 20K. The structure of Arfaptin-Rac1 was solved by molecular replacement and showed that one molecule of Rac1 binds to a dimer of Arfaptin as predicted from binding measurements, with Rac1 sitting roughly at the midpoint of the Arfaptin dimer (Figure 3). As the crystals initially took 3 months to grow at 18 °C, we monitored the quality and stability of the complex over this period of time using the technique of dynamic light scattering (DLS). DLS can be used to characterize protein homogeneity, size, and thermal stability by measuring the molecular diffusion constant, size, and mono- or polydispersity of particles. Protein solutions of Rac1 and Arfaptin and the mixed complex were analyzed by DLS on a weekly basis. Data from these measurements are listed in Table 2, showing the different behavior of the apo proteins and holo
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Crystal Growth & Design, Vol. 7, No. 11, 2007 2215 Table 2. Dynamic Light Scattering Data from Solutions of Rac1, Arfaptin, and Rac1–Arfaptin Complexa
protein
Dt (× 10-9 cm2 s-1)
Rh (nm)
mol wt (kD)
base line
SOS error
Cp/Rh (%)
comments crystallizable
Rac-1 Rac-2 Rac-3 Arfaptin-1 Arfaptin-2 Arfaptin-3 complex-1 complex-2
793.3 828.6 605.8 502.6 432.3 451.4 416.5 416.6
2.5 2.3 3.5 4.2 4.2 5.3 4.8 4.7
27.8 22.2 60.8 92.1 91.7 167.1 126.7 125.3
1.001 1.001 1.002 0.999 1.000 1.006 0.998 0.999
0.857 7.500 1.380 0.941 0.676 10.800 1.880 1.430
13.2 20.1 22.8 9.7 14.6 43.6 6.2 7.7
yes yes no yes yes no yes yes
a
All measurements were performed using a DanaPro MS801 (Wyatt Tech).
Figure 4. Multiple sequence alignment of 27 species of Rb and Rb-related P107 and P130 proteins. The conservation is based on sequence identity; residues identical across these 27 sequences are in blue and the degree of similarity is represented by the degree of blueness. The N-terminal is underlined in white, the domain A in red, the domain B in blue, the C-terminal in grey, and the spacer in cream. Species are selected in the following: P06400, P33568, NP_000312, NP_033055, AAD13990, AAB18279-p107, Q64701-p107, T01173, AAA36397-p107, NP_005602-p130, O055081-p130, AAB48991-p130, CAA09736, AAF61377, BAA76477, AAF79146, BAA88690, AAF67147, CAA51019, CAA70428, A44879, AF230739, AAG12717, AAF97520, I49328-p107, Q08999-p130, Q64700-p130.
solutions and whether or not crystals could be obtained from them. Before both the apo and holo structures were solved, it was curious that the apparent molecular weights of the complex (Arfaptin:Rac 2:1), which were measured as 117–126 kD, were significantly higher than the calculated mass of 72 kD (apo-Arfaptin shows a similar phenomenon as well). This discrepancy may be due to an irregular molecular shape or the presence of too many flexible or disordered parts on the molecular surface. After the structure of Arfaptin was solved, it was obvious that the much higher apparent molecular weight was caused by its elongated shape. Now we can use light scattering technique to measure the absolute molecular weight to characterize the real situation in protein solution in just over 1 hour. The multiangle laser light scattering detector combining with HPLC (DAWN, Wyatt Technology) can measure the absolute molecular weight, size, and conformation to characterize its homogeneity, polydispersity, and oligomeric states in a more accurate way than singleangle detector, and it could ensure that the new equipment
would give more guides to direct the successful protein crystallization in the future. 2. Using Information from Bioinformatics and Proteomics To Generate Favorable Crystallization Fragments Rb plays important roles in regulating the cell cycle, apoptosis, and differentiation, and all of these activities are pertinent to its role as a tumor suppressor. Mutation of Rb or disruption of its regulatory partners is involved in most human cancers. In the eukaryotic cell cycle, Rb negatively regulates the G1-S transition by binding to the E2F transcription factors, and its repression is relieved after phosphorylation by specific cyclin/CDKs and release of E2F binding. To understand the regulation of the E2F by Rb, we have determined the crystal structure of Rb complexed to E2F peptide.11 Human Rb has 928 residues and can be divided into four main domains (N-terminal, A, B, and C-terminal) using limited proteolysis by trypsin, chymotrypsin, and subtilisin.12 The N-terminal is largely uncharacterized and its function is
2216 Crystal Growth & Design, Vol. 7, No. 11, 2007
Figure 5. Structure of pRb–E2F complex in two orthogonal views. The helices of the A domain are shown as red cylinders and those of the B domain are shown as blue cylinders. The main chain trace of E2F is shown as yellow worm.
Reviews
secondary structure, and these are linked by a flexible prolinerich spacer. The crystal structure of domains A and B of Rb complexed with a peptide of papilloma virus E7 was the first Rb structure to be solved.13 We have tried to crystallize various constructs of Rb that include both the A and B domains and various lengths of C domain, which contains the important phosphorylation sites, but all these constructs resisted crystallization. Following this, a multiple sequence alignment was used to try to predict a favorable region for crystallization. The alignment of 27 species of Rb and the Rb-related family members p107 and p130 proteins clearly shows the four main domains and an unconserved spacer sequences between domain A and B (Figure 4). Domains A and B are the most highly conserved regions and may be the best candidates for crystallization. The Rb-AB construct was engineered to contain a prescission protease site at the N-terminus and two thrombin sites inserted at both ends of the flexible A–B linker. After the linker has been removed, domains A and B can still be associated together noncovalently.14 Our aim was to crystallize Rb in complex with E2F. Various constructs of E2F1 were tested without success in the crystallization trials; however, particular attention was paid to a highly conserved region in the E2F transactivation area, where nine residues in E2F (409–426) are conserved across E2Fs from all animal species. These residues bind Rb directly and were selected for crystallization trials. Crystals of the Rb–E2F (409–426) complex grew in a platelike fashion from precipitates using the hanging drop method at 4 °C. The reservoir solution contained 0.14 M Na citrate, 26% PEG400, 4% n-propanol, and 0.1 M TRIS at pH 7.8. Repeated attempts at data collection from flash-cooled crystals using synchrotron X-ray sources were frustrated by very high crystal mosaicity of up to 5° with poor data reduction statistics. Furthermore, intensive attempts to change the crystal morphology failed. The problem was eventually overcome by using the microfocus diffractometer on station ID13 at the European Synchrotron Radiation Facility (ESRF) using a 10 µm aperture. Data were collected from four separate and widely spaced volumes of a single crystal in order to minimize radiation damage. The structure was solved by molecular replacement to reveal that the packing of the A and B domains generates a groove into which E2F peptide binds in a largely extended manner (shown in Figure 5). We have also tried to crystallize various constructs of Rb that include segments of the C domain, which contains phosphorylation sites important for regulation. Unfortunately, these attempts failed, probably because the C domain is highly flexible. Recently, Pavletich and his colleagues successfully crystallized Rb-C (829–874) complexed to E2F1-DP1 heterodimer after using limited proteolysis to define the secondary structure boundaries of the E2F-DP heterodimer.15 This work showed that this part of the C domain becomes structured on binding to E2F-DP. 3. Optimizing Crystallization Conditions from NMR and Other Information
Figure 6. Cofactor models of SET domain proteins. (A) AdoMet (Sadenosyl-L-methionine), (B) AdoHcy (S-adenosyl-L-homecystine).
unknown; the C-terminal has the least defined secondary structure. The domains A and B have much more defined
In eukaryotic cells, DNA is packed by histone proteins into a higher-ordered structure termed chromatin. The basic unit of chromatin, the nucleosome, consists of an octomer of histone proteins that form a core to accommodate two turns (146 base pairs) of double-stranded DNA. The N-terminals of the four different histone proteins (H2A, H2B, H3, and H4) protrude out from the core structure and interact with specific binding partners. The evolutionarily conserved SET domain is a motif
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Crystal Growth & Design, Vol. 7, No. 11, 2007 2217
Figure 7. Selected regions from the 1H{13C}-HSQC spectra of complexes comprising SET7/9 (52–366) and a 20 residue peptide of histone H3, specifically 13C/15N-labeled at lysine-4. Spectra were recorded at 25 °C on a Varian Inova spectrometer operating at a1H frequency of 600 MHz. (A) Prior to addition of AdoMet, the peaks are shown arising from distinct Ce moieties of Lysine-4. (B) After incubation overnight with the methylated lysine-4.
of about 130 residues and is present in many eukaryotic chromatin associated proteins. All SET-domains have been found to have histone lysine methyltransferase activity. Methylation of lysine residues in histone tails can be used for generating epigenetic marks and disruption of the normal epigenetic process can cause disease. Histone methyltransferases are responsible for methyl transfer from S-adenosylmethionine (AdoMet) to the histone lysine side chain nitrogen. To better understand the biological role of the enzyme, we studied the structure of SET7/9 in complex with H3 (1–9) peptide and AdoMet. Human methyltransferase SET7/9 methylates histone H3 lysine 4 (K4). The purified full length protein (1–366) was grown into small needlelike crystals, but they did not diffract. A proteolytic digest of this fragment was performed, which produced new constructs (residues 52–344 and 52–366) that
were tested in crystallization trials. The apo-protein SET7/9 (construct 52–344) was then crystallized in hanging drops equilibrated with reservoir solution containing 0.2 M magnesium formate and 25% PEG3350 at 18 °C and diffracted to 2.1 Å spacing. Following this, the structure of SeMet SET7/9 (52–344) was determined using MAD experiments.16 The shortened construct (52–344) was still able to bind the substrate and cofactor, even with removal of the last 22 residues at the C terminus, but activity measurements showed that it had lost its catalytic ability. Therefore, for crystallization of the ternary complex, a construct encompassing residues 52–366 was used instead. In SET methylation, the cofactor AdoMet is required as the methyl-group donor; however, AdoMet is not stable over crystallization time periods because of the sulfonium center, which leads to the decomposition and/or epimerization
2218 Crystal Growth & Design, Vol. 7, No. 11, 2007
Reviews
complex solution. Also, we have used information from bioinformatics and proteomics to engineer favorable constructs and incorporated available information from NMR, chemistry, and other sources for optimizing crystallization in order to obtain crystals of diffraction quality at higher resolution.
References
Figure 8. Structure of the SET7/9 ternary complex in two orthogonal views, shown in ribbon presentation. The N-terminal domain is colored pink, the SET domain is blue, and the C-terminal is grey. The H3 peptide is colored green and the AdoHcy (S-adenosyl-L-homecystine) cofactor is yellow.
of AdoMet. Thus, the more stable S-adenosyl-L-homecystine (AdoHcy) was used as the cofactor (shown in Figure 6). The crystallization trial of SET7/9 (52–366)-H3 (1–9)-AdoHcy and SET7/9 (52–366)-H3 (1–19)-AdoHcy did not give any crystals. In addition to the crystallization trials, a series of NMR experiments were performed examining any potential ordering of SET7/9 upon binding of histone peptides. These studies (using a shortened construct 108–366, which was less susceptible to degradation) revealed that the H3 peptide containing monomethylated K4 was better ordered in complex with SET7/9 than the unmodified peptide (Figure 7). The relative intensities of the new peaks appearing on methylation (at 2.87, 51.4 and 2.63, 42.0 ppm) indicate that the methylated peptide is more stable bound than the unmethylated substrate. So, monomethyl K4 H3 peptide was used as a substrate for crystallization: the protein was incubated with a 2-fold molar excess of monomethyl K4 H3 (1–9) and AdoHcy. The hanging drops were prepared by mixing equal volumes of protein complex with reservoir solution containing 22% PEG3350 and 0.1 M TRIS, pH 7.8, at 18 °C. Crystals were readily grown overnight. The crystals of the ternary complex were well-ordered and diffracted in house to at least 1.7 Å spacing and the structure was solved by molecular replacement (shown in Figure 8).17 The C-terminal segment, the AdoHcy cofactor, and most of the substrate peptide are welldefined in the electron density maps, as are all important residues around the active site. Furthermore, using these crystals as seeds, we grew crystals in the solutions of ternary complexes with substrates of nonmodified H3 (1–9) and H3 (1–19) and monomethyl K4 H3 (1–19). It is interesting to note that the complex of histone methyltransferases with a monomethyl histone peptide has aided crystallization in other SET domain proteins. For example, the crystal of ternary complex of Pr-Set7 with monomethyl K20 H4 (17–25) and AdoHcy diffracted up to 1.4 Å spacing using synchrotron radiation.18 Summary This is a review of our experimental trials designed to optimize protein complexes for crystal growth over the last five years. We illustrate how to maximize complex formation using biophysical techniques and how to monitor the stability of the
(1) Auerbach-Nevo, T.; Zarivach, R.; Peretz, M.; Yonath, A. Reproducible growth of well diffracting ribosomal crystals. Acta Crystallogr., Sect. D 2005, 61, 713–719. (2) Yamada, Y.; Inoue, M.; Shiba, T.; Kawasaki, M.; Kato, R.; Nakayama, K.; Wakatsuki, S. Structure determination of GGA-GAE and γ1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic. Acta Crystallogr., Sect. D 2005, 61, 731–736. (3) Ménez, R.; Housden, N. G.; Harrison, S.; Jolivet-Reynaud, C.; Gore, M. G.; Stura, E. A. Different crystal packing in Fab-protein L semidisordered peptide complex. Acta Crystallogr., Sect. D 2005, 61, 744– 749. (4) Rossmann, M. G.; Arisaka, F.; Battisti, A. J.; Bowman, V. D.; Chipman, P. R.; Fokine, A.; Hafenstein, S.; Kanamaru, S.; Kostyuchenko, V. A.; Mesyanzhinov, V. V.; Shneider, M. M.; Morais, M. C.; Leiman, P. G.; Palermo, L. M.; Parrish, C. R.; Xiao, C. From structure of the complex to understanding of the biology. Acta Crystallogr., Sect. D 2007, 63, 9–16. (5) Dafforn, T. R. So how do you know you have a macromolecular complex. Acta Crystallogr., Sect. D 2007, 63, 17–25. (6) Ladbury, J. E. Measurement of the formation of complexes in tyrosine kinase-mediated signal transduction. Acta Crystallogr., Sect. D 2007, 63, 26–31. (7) Hassell, A. M.; An, G.; Bledsoe, R. K.; Bynum, R. K.; Carter, H. L.; Deng, S.-J. J.; Gampe, R. T.; Grisard, T. E.; Madauss, K. P.; Nolte, R. T.; Rocque, W. J.; Wang, L.; WeaverK. L.; Williams, S. P.; Wisely, G. B.; Xu, R.; Shewchuk, L. M. Crystallization of protein-ligand complexes. Acta Crystallogr., Sect. D 2007, 63, 72–79. (8) Van Aelst, L.; Joneson, T.; Bar-Sagi, D. Identification of a novel Rac1interacting protein involved in membrane ruffling. EMBO J. 1996, 15, 3778–3786. (9) Kanoh, H.; Williger, B.; Exton, J. H. Arfaptin 1,a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes. J. Biol. Chem. 1997, 272, 5421–5429. (10) Tarricone, C.; Xiao, B.; Justin, N.; Walker, P. A.; Rittinger, K.; Gamblin, S. J.; Smerdon, S. J. The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways. Nature 2001, 411, 215–9. (11) Xiao, B.; Spencer, J.; Clements, A.; Ali-Khan, N.; Burghammer, M.; Perrakis, A.; Gamblin, S. J. Crystal structure of the retinoblastoma tumour suppressor protein:E2F peptide complex and implications for the action of the papillomavirus E7 oncoprotein. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 2362–8. (12) Hensey, C. E.; Hong, F.; Durfee, T.; Qian, Y. W.; Lee, E. Y.; Lee, W. H. Identification of discrete structural domains in the retinoblastoma protein. J. Biol. Chem. 1994, 269, 1380–1387. (13) Lee, J. O.; Russo, A. A.; Pavletich, N. P. Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV E7. Nature 1998, 391, 859–865. (14) Chow, K. N.; Dean, D. C. Domains A and B in the Rb pocket interact to form a transcriptional repressor motif. Mol. Cell. Biol. 1996, 16, 4862–4868. (15) Rubin, S. M.; Gall, A. L.; Zheng, N.; Pavletich, N. P. Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release. Cell 2005, 123, 1093–1106. (16) Wilson, J. R.; Jing, C.; Walker, P. A.; Martin, S. R.; Howell, S. A.; Blackburn, G. M.; Gamblin, S. J.; Xiao, B. Crystal structure and functional analysis of the histone methyltransferases SET7/9. Cell 2002, 111, 105–115. (17) Xiao, B.; Jing, C.; Wilson, J. R.; Walker, P. A.; Vasisht, N.; Kelly, G.; Howell, S.; Taylor, I.; Blackburn, G. M.; Gamblin, S. J. Crystal structure of a ternary complex of the human Histone Methyltransferase SET7/9. Nature 2003, 421, 652–6. (18) Xiao, B.; Jing, C.; Kelly, G.; Walker, P. A.; Muskett, F. W.; Frenkiel, T. A.; Martin, S. R.; Sarma, K.; Reinberg, D.; Gamblin, S. J.; Wilson, J. R. Specificity and mechanism of the histone methyltransferase PrSet7. Genes DeV. 2005, 19, 1444–1454.
CG7007039
