Figures
Abstract
SUV39H1, the first identified histone lysine methyltransferase in human, is involved in chromatin modification and gene regulation. SUV39H1 contains a chromodomain in its N-terminus, which potentially plays a role in methyl-lysine recognition and SUV39H1 targeting. In this study, the structure of the chromodomain of human SUV39H1 was determined by X-ray crystallography. The SUV39H1 chromodomain displays a generally conserved structure fold compared with other solved chromodomains. However, different from other chromodomains, the SUV39H1 chromodomain possesses a much longer helix at its C-terminus. Furthermore, the SUV39H1 chromodomain was shown to recognize histone H3K9me2/3 specifically.
Citation: Wang T, Xu C, Liu Y, Fan K, Li Z, Sun X, et al. (2012) Crystal Structure of the Human SUV39H1 Chromodomain and Its Recognition of Histone H3K9me2/3. PLoS ONE 7(12): e52977. https://doi.org/10.1371/journal.pone.0052977
Editor: Esteban Ballestar, Bellvitge Biomedical Research Institute (IDIBELL), Spain
Received: May 21, 2012; Accepted: November 22, 2012; Published: December 28, 2012
Copyright: © 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Basic Research Program of China (973 Program), grant number 2009CB918804, the Knowledge Innovation Program of the Chinese Academy of Science, grant number KSCX2-EW-Q-4, and the Chinese National Natural Science Foundations, grant number 31270780. This work was also supported by the Structural Genomics Consortium, a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institute, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust to JM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that they received funding from commercial sources - GlaxoSmithKline and Merck & Co., Inc. However, this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Introduction
In eukaryote, histone modifications play an important role in regulating gene expression in the native chromatin context. The amino-terminal tails of nucleosomal histones, protruding away from the nucleosome core, are amenable to several forms of posttranslational modifications such as methylation, acetylation, phosphorylation, ADP ribosylation, and ubiquitination [1]. As an abundant epigenetic modification, histone lysine methylation is essential for the organization and function of chromatin. The methylation patterns have been associated with distinct chromatin states and are proposed to be the major epigenetic marks that could extend the genetic code by regulating the chromatin structure in a heritable manner [2].
The human SUV39H1, a histone H3K9 methyltransfearse, is the first histone lysine methyltransferase (HMT) identified, which, together with SUV39H2, are the mammalian homologs of Drosophila Su(var)3-9 and Schizosaccharomyces pombe Clr4. A direct consequence of this modification is the creation of a high-affinity binding site for heterochromatin protein 1 (HP1), which together with other proteins induces chromatin packaging and gene silencing [3], [4]. In addition to initiating the formation of large heterochromatin regions, SUV39H1 is also involved in repressing the transcription of specific genes. It interacts with DNA-binding proteins involved in the leukemogenesis such as AML1 and PML-RARα, promoting the silencing of their target genes [5], [6].
SUV39H1 possesses a SET domain at its C-terminus, which performs the catalytic activity, and a chromodomain at its N-terminus. The chromodomain is a conserved motif containing about 50 amino acids and is identified as a module to target proteins to specific chromosomal loci [7]. The chromodomain family displays a broad range of activities, including methyl-lysine histone, DNA and RNA binding [8]–[10]. It was revealed that the chromodomain of SUV39H1 is essential for the catalytic activity of SUV39H1 [11]. Mutation and deletion of the chromodomain of SUV39H1 impaired its enzyme activity in spite of the presence of an intact catalytic SET domain [11]. Presumably binding methylated histones by the chromodomain of SUV39H1 plays a critical role in targeting the catalytic activity of SUV39H1.
Here we report the crystal structure of the chromodomain of human SUV39H1. The structure exhibits a fold similar to other solved chromodomain structures. Furthermore, we identify that the SUV39H1 chromodomain is able to recognize H3K9me2/3 specifically by fluorescence polarization binding assay.
Results
The SUV39H1 Chromodomain Exhibits Moderate Sequence Similarity and Conserved Structure Compared with other Chromodomains
Sequence alignment analysis showed that the human SUV39H1 chromodomain displays moderate sequence identity and similarity in comparison with other chromodomains (Fig. 1A). The SUV39H1 chromodomain shares about 30% sequence identity and about 40% sequence similarity with other chromodomains, including human and Drosophila HP1 and Polycomb proteins, which are the founding members of chromodomains and whose structures have been determined [8], [12]–[15]. Nevertheless, the SUV39H1 chromodomain shows highly conserved structure features shared in the chromodomain family, including the aromatic cage residues that implies their potential ability in methyl-lysine histone binding (Fig. 1A).
A: Alignment of chromodomain sequences of human SUV39H1, human MPP8, Drosophila HP1, Drosophila polycomb protein and human chromobox homologs CBX3. HsSUV39H1, human SUV39H1 chromodomain; HsMPP8, human MPP8 chromodomain; DmHP1, Drosophila melanogaster HP1 chromodomain; DmPolycomb, Drosophila melanogaster polycomb protein chromodomain; HsCBX3, human chromobox homolog 3, HP1γ. Secondary structures of the SUV39H1 chromodomain are labeled at the top and the aromatic cage residues are marked by as well. B: The crystallographic asymmetric unit of human SUV39H1 chromodomain, colored in cyan, green and yellow, respectively. C: The ribbon diagram of human SUV39H1 chromodomain monomer colored in cyan and secondary structures are labeled.
Overall Structure of SUV39H1 Chromodomain
The crystal structure of the human SUV39H1 chromodomain (aa 44–106) was determined at a resolution of 2.2 Å, and deposited in the Protein Data Bank with an accession number 3MTS. The human SUV39H1 chromodomain, which contains 3 independent molecules in an asymmetric unit, adopts a canonical chromodomain architecture (Fig. 1B/C). However, it exists as a monomer in solution. The fold is composed of an N-terminal SH3-like β-barrel, followed by a long C-terminal helix α1(residues 82–100). The β-barrel consists of a sheet with three antiparalled strands β1 (residues 45–53), β2 (residues 58–64) and β3 (residues 73–76).
Structural Comparison between Human SUV39H1 Chromodomain and other Chromodomain Family Members
Several structural homologs of the SUV39H1 chromodomain were identified by DALI21 (http://ekhidna.biocenter.helsinki.fi/dali_server). The best matched structure identified in the PDB library is that of the chromodomain of MPP8 (PDB code 3R93) [14], with Z = 9.6, RMSD = 1.2 Å, and a sequence identity of 31%. The Z-score and RMSD between the SUV39H1 chromodomain and the Drosophila HP1 chromodomain (PDB code 1KNE) are 9.4 and 1.0 Å, respectively, which demonstrates structure conservation between the two structures. Therefore, except for the longer helix α1, the human SUV39H1 chromodomain (Fig. 2A) is structurally very similar to other determined chromodomains (Fig. 2B/C). Interestingly, the chromodomain of the human SUV39H1 we crystallized lacks the first aromatic residue F43 of the aromatic cage. Thus the two aromatic residues, W64 and Y67, in the loop between β2 and β3 form a partial aromatic cage, which is a conserved structural feature among chromodomain proteins and other Royal family members [16]. This may implicate that the cage structural element is not essential for fold stability. The aromatic cage has been widely utilized for recognizing methylated lysine or arginine of proteins, such as the tudor domain of SGF29, which binds histone H3K4me2/3 and targets the SAGA complex [17], the tudor domain of SND1, which binds arginine methylated PIWI proteins and recruits its associated RNA cleavage activity [18], the MBT domain of L3MBTL1/2, which recognizes lower methyated lysine histones [19], [20], the chromo barrel domain of Eaf3, which is a subunit of the NuA4 histone acetyltransferase complex and recognizes methylated H3K36 [21] and the WD40 domain of EED, which is a histone H3K27 reader and a compoent of PRC2 complex [22], [23]. In the next section, we are going to discuss more regarding the histone binding ability of the partial chromodomain of SUV39H1.
B: human MPP8 chromodomain (PDB ID: 3R93) C: Drosophila HP1 chromodomain (PDB ID: 1KNE).
Another conserved structural feature of the chromdomain family is that it contains a hydrophobic core consisting of the residues V45, L48, Y60, V62, W64, L80, I85 and L86. All of these residues are also conserved in the chromodomain family (Fig. 1A). Interestingly, the potential peptide binding groove formed by the SUV39H1 chromdomain β sheet is reminiscent of the binding grove identified in the PTB and PDZ domains [24]. All these observations revealed that the human SUV39H1 chromodomain adopts an overall structure similar to that of the other chromodomain family members and contains conserved residues, which form a compact core with a aromatic cage crucial for binding methyllysine [16].
SUV39H1 Chromodomain Recognizes H3K9me3 by Fluorescence Polarization Assays
Because the chromodomain we crystallized contains an incomplete aromatic cage, we next examine if the deletion of the first aromatic residue F43 in SUV39H1 affects its methyl-lysine histone binding. We purified two SUV39H1 constructs including the one used for crystallization (aa 44–106) and the one containing a complete aromatic cage (aa 42–100). By means of fluorescence polarization assay, we found that the partial chromodomain does not show detectable binding to any histone H3K9 peptides (Fig. 3A, Table 1). The importance of a complete aromatic cage has been show previously for CDYL, which lacks the first aromatic residue and lacks histone binding as well [25]. Not surprisingly, the complete chromodomain of SUV39H1 specifically binds to histone H3K9me2/3 with a Kd of 20±4 µM for H3K9me3, and a Kd of 29±12 µM for H3K9me2 (Fig. 3B, Table1). It does not show detectable binding to histone H3K9me0/1 (Fig. 3B, Table 1). In addition, the SUV39H1 chromodomain just shows very weak binding to histone H3K27me3 (Fig. 3B, Table 1). Taken together, the SUV39H1 chromodomain specifically recognizes histone H3K9me2/3.
Diagrams of different H3 peptides are indicated by different symbols. A: Kinetic analysis of interactions between truncated SUV39H1 chromodomain (aa 44–106) and H3K9me3/2/1/0 peptide. B: Kinetic analysis of interactions between complete SUV39H1 chromodomain (aa 42–100) and H3K9me3/2/1/0 or H3K27me3 peptide.
Discussion
SUV39H1 is the first identified histone lysine methyltransferase (HMT) in human [26], [27]. It catalyzes di- and tri-methylation of lysine 9 of histone H3, which are related to chromatin packaging and gene silencing [3], [4]. SUV39H1 possesses a chromodomain at its N-terminus and a SET domain at its C-terminus. Although the SET domain performs the catalytic activity, the chromodomain of SUV39H1 is vital for the catalytic activity of SUV39H1 [11].
In this study we determined the 3D structure of the chromodomain of human SUV39H1. It exhibits a three-dimensional fold similar to that of the determined structures of other chromodomains. However, it possesses a much longer helix at its C-terminus, which is different from other chromodomains. Additionally, we showed that the SUV39H1 chromodomain specifically binds to H3K9me2/3. The chromodomains of HP1 and MPP8 preferentially bind to H3K9me3 [14]. We also confirmed that the SUV39H1 chromodomain also binds H3K9me3 and H3K9me2, albeit the latter binds about 1.5 fold weaker. However, no strong interactions between the chromodomain of human SUV39H1 and H3K27me3 were observed, which indicates that the chromodomain of human SUV39H1 would bind H3K9me2/3 specifically. Residues Q5-S10 (QTARKS) in the H3K9 peptide are critical for the interactions between the chromodomains and the H3K9 peptide. H3K9 and H3K27 share the sequence of ARKS, however, the sequence in H3K27 is KA instead of QT preceding the conserved ARKS motif. This difference was identified to be important for distinguishing H3K9 from H3K27 [14]. Therefore, our binding results show that the SUV39H1 chromodomain specifically recognizes the histone H3K9me2/3 mark, and the detailed molecular mechanism of how the SUV39H1 chromodomain specifically recognizes histone H3K9me2/3 warrant future complex structure determination.
It has been reported that the chromodomain of human SUV39H1 is essential for the protein’s catalytic activity. Deletion of the chromodomain or point mutation of the conserved amino acids, W64A or Y67A, of the chromodomain in SUV39H1 impaired the enzyme activity even if the catalytic SET domain is intact [11]. Consistently, it has been show that the chromodomain of Clr4, which is a Schizosaccharomyces pombe homolog of human SUV39H1, binds specifically to H3K9me and is responsible for its H3K9 methylation activity targeting and the spreading of heterochromatin [11], [28]. It is tempting to speculate that the chromodomain of SUV39H1 would play a similar role to that of the Clr4 chromodomain. Sequence alignment and structural comparison demonstrated that the human SUV39H1 chromodomain is similar to the Drosophila HP1 chromodomain, which was the first chromodomain characterized structurally at an atomic level and provided insight into its function as a methylated histone lysine binding domain [15], [29]. The methylated ligand is coordinated by three aromatic residues that form an open “cage”, which is partially hydrophobic around the moiety. The sequence alignment revealed that there are approximately 29% sequence identities and 42% similarity between the human SUV39H1 chromodomain and Drosophila HP1 chromodomains (Fig. 1A). Additionally, their 3D structures are also similar to each other. Especially, the key residues responsible for the recognition of H3K9me3, W45 and Y48 in Drosophila HP1 chromodomain, are conserved in the two sequences. The residues are located in the similar region of the structures and the side chains protrude from the backbone in a similar orientation (Fig. 1A & Fig. 4). Therefore, the corresponding residues in human SUV39H1 chromodomain, W64 and Y67, are supposed to be also important for methylated histone lysine binding. Sequence and structure similarity between the two chromodomains, and especially the conservation of the key residues involved in the “cage”, together suggest that they may interact with histone H3K9me3 in a similar way.
The structures of human SUV39H1 and Drosophila melanogaster HP1 (PDB: 1KNE) chromodomains are aligned and shown in magenta and cyan, respectively. Y24, W45 and Y48 of Drosophila melanogaster HP1 chromodomain that are critical for H3K9me3 binding are shown as sticks in blue. The corresponding residues, W64 and Y67 of human SUV39H1 chromodomain, are shown as sticks in red. H3K9me3 peptide is shown in yellow with trimethylated lysine 9 shown as sticks.
Materials and Methods
Protein Expression and Purification
The human SUV39H1 chromodomain (residues 44–106 or 42–100) was subcloned into pET28a-MHL vector. SUV39H1 chromodomain for crystallization and binding assays are the fragments from residue 44 to 106 and from residue 42 to 100, respectively. The recombinant protein was over-expressed at 18°C as an N-terminal His6-tagged protein in E. coli BL21 (DE3) Codon plus RIL (Stratagene) and was purified by HiTrap Ni column [30]. The obtained proteins were further purified on a HiLoad 16/60 Superdex 200 prep-grade column. The final sample for crystallization contained 10 mg/ml SUV39H1 chromodomain and 3.5 M Na Formate, 0.1 M Bis-Tris Propane (pH 7.0) for crystallization.
Data Collection, Structure Determination and Refinement
Diffraction data were collected at 100 K on beamline 23-ID-B (GM/CA-CAT, Advanced Photon Source, Argonne National Laboratory) using a MARMOSAIC 300 CCD detector. The crystal belonged to space group R32, with unit cell parameters a = b = 99.5, c = 118.3 Å, and diffracted to 2.2 Å resolution. The data were integrated and scaled using the HKL2000 software package [31]. The structure was solved by molecular replacement using the crystal structure of the chromo domain of HP1 from Drosophila melanogaster (PDB 1KNE) as a search model and the program Phaser as implemented in the Phenix program suite [32], [33]. Following several alternate cycles of restrained refinement and manual rebuilding using COOT, the improved model revealed clear electron densities allowing placement of ordered solvent molecules [34]. All refinement steps were performed using REFMAC in the CCP4 program suite [35], [36]. During the final cycles of model building, TLS parameterization was included in the refinement which comprised three protein chains and solvent molecules [37], [38]. Data collection and refinement statistics are summarized in Table 2. The stereochemical quality of the final model was validated by PROCHECK online (http://nihserver. mbi.ucla.edu/SAVES_3/).
Fluorescence Polarization
All peptides used for fluorescence polarization measurements were synthesized by Tufts University Core Services. The assay was performed in 10 µL at a constant fluorescence labeled-peptide concentration of 40 nM and increasing amounts of SUV39H1 (residues 42–100 or 44–106) at concentrations ranging from low to high micromolar in a buffer of 20 mM Tris • HCl, pH 8.5 or 7.5, 150 mM NaCl, 1 mM DTT, and 0.01%Tween-X-100. The assay was performed in 384-well plates, using a Synergy 2 microplate reader (BioTek). An excitation wave length of 485 nm and an emission wave length of 528 nm were used. The data were corrected for background of the free-labeled peptides. To determine the Kd values, the data were fit to a hyperbolic function using Sigma Plot software (Systat Software, Inc.).
Author Contributions
Conceived and designed the experiments: TW CX JM XT. Performed the experiments: TW CX Y. Liu KF ZL XS HO Y. Li FM. Analyzed the data: TW CX Y. Liu KF. Contributed reagents/materials/analysis tools: XZ JZ. Wrote the paper: TW CX JM XT.
References
- 1. Kouzarides T (2007) Chromatin modifications and their function. Cell 128: 693.
- 2. Volkel P, Angrand PO (2007) The control of histone lysine methylation in epigenetic regulation. Biochimie 89: 1–20.
- 3. Melcher M, Schmid M, Aagaard L, Selenko P, Laible G, et al. (2000) Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression. Molecular and Cellular Biology 20: 3728–3741.
- 4. Lachner M, O'Carroll D, Rea S, Mechtler K, Jenuwein T (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410: 116–120.
- 5. Chakraborty S, Sinha KK, Senyuk V, Nucifora G (2003) SUV39H1 interacts with AML1 and abrogates AML1 transactivity. AML1 is methylated in vivo. Oncogene 22: 5229–5237.
- 6. Carbone R, Botrugno OA, Ronzoni S, Insinga A, Di Croce L, et al. (2006) Recruitment of the histone methyltransferase SUV39H1 and its role in the oncogenic properties of the leukemia-associated PML-retinoic acid receptor fusion protein. Molecular and Cellular Biology 26: 1288–1296.
- 7. Brehm A, Tufteland KR, Aasland R, Becker PB (2004) The many colours of chromodomains. Bioessays 26: 133–140.
- 8. Kaustov L, Ouyang H, Amaya M, Lemak A, Nady N, et al. (2011) Recognition and specificity determinants of the human cbx chromodomains. J Biol Chem 286: 521–529.
- 9. Bernstein E, Duncan EM, Masui O, Gil J, Heard E, et al. (2006) Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin. Mol Cell Biol 26: 2560–2569.
- 10. Kim D, Blus BJ, Chandra V, Huang P, Rastinejad F, et al. (2010) Corecognition of DNA and a methylated histone tail by the MSL3 chromodomain. Nat Struct Mol Biol 17: 1027–1029.
- 11. Chin HG, Patnaik D, Esteve PO, Jacobsen SE, Pradhan S (2006) Catalytic properties and kinetic mechanism of human recombinant lys-9 histone H3 methyltransferase SUV39H1: Participation of the chromodomain in enzymatic catalysis. Biochemistry 45: 3272–3284.
- 12. Min JR, Zhang Y, Xu RM (2003) Structural basis for specific binding of polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev 17: 1823–1828.
- 13. Fischle W, Wang Y, Jacobs SA, Kim Y, Allis CD, et al. (2003) Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev 17: 1870–1881.
- 14. Li J, Li Z, Ruan J, Xu C, Tong Y, et al. (2011) Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9. PLoS One 6: e25104.
- 15. Nielsen PR, Nietlispach D, Mott HR, Callaghan J, Bannister A, et al. (2002) Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416: 103–107.
- 16. Adams-Cioaba MA, Min JR (2009) Structure and function of histone methylation binding proteins. Biochemistry and Cell Biology-Biochimie Et Biologie Cellulaire 87: 93–105.
- 17. Bian C, Xu C, Ruan J, Lee KK, Burke TL, et al. (2011) Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation. EMBO J 30: 2829–2842.
- 18. Liu K, Chen C, Guo Y, Lam R, Bian C, et al. (2010) Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain. Proc Natl Acad Sci U S A 107: 18398–18403.
- 19. Min JR, Allali-Hassani A, Nady N, Qi C, Hui OY, et al. (2007) L3MBTL1 recognition of mono- and dimethylated histones. Nat Struct Mol Biol 14: 1229–1230.
- 20. Guo Y, Nady N, Qi C, Allali-Hassani A, Zhu H, et al. (2009) Methylation-state-specific recognition of histones by the MBT repeat protein L3MBTL2. Nucleic Acids Res 37: 2204–2210.
- 21. Xu C, Cui G, Botuyan MV, Mer G (2008) Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S. Structure 16: 1740–1750.
- 22. Xu C, Bian C, Yang W, Galka M, Ouyang H, et al. (2010) Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2). Proc Natl Acad Sci U S A 107: 19266–19271.
- 23.
Nayak V, Xu C, Min J (2011) Composition, recruitment and regulation of the PRC2 complex. Nucleus 2.
- 24. Kuriyan J, Cowburn D (1997) Modular peptide recognition domains in eukaryotic signaling. Annual Review of Biophysics and Biomolecular Structure 26: 259–288.
- 25. Fischle W, Franz H, Jacobs SA, Allis CD, Khorasanizadeh S (2008) Specificity of the chromodomain Y chromosome family of chromodomains for lysine-methylated ARK(S/T) motifs. J Biol Chem 283: 19626–19635.
- 26. Aagaard L, Laible G, Selenko P, Schmid M, Dorn R, et al. (1999) Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3–9 encode centromere-associated proteins which complex with the heterochromatin component M31. Embo Journal 18: 1923–1938.
- 27. Rea S, Eisenhaber F, O'Carroll D, Strahl BD, Sun ZW, et al. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: 593–599.
- 28. Zhang K, Mosch K, Fischle W, Grewal SI (2008) Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin. Nat Struct Mol Biol 15: 381–388.
- 29. Jacobs SA, Khorasanizadeh S (2002) Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science's STKE 295: 2080.
- 30. Xu C, Bian C, Lam R, Dong A, Min J (2011) The structural basis for selective binding of non-methylated CpG islands by the CFP1 CXXC domain. Nat Commun 2: 227.
- 31. Otwinowski Z, Minor W (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode. Meth Enzymol 276: 307–206.
- 32. Mccoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, et al. (2007) Phaser crystallographic software. Journal of Applied Crystallography 40: 658–674.
- 33. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D-Biological Crystallography 66: 213–221.
- 34. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallographica Section D-Biological Crystallography 60: 2126–2132.
- 35. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallographica Section D-Biological Crystallography 53: 240–255.
- 36. Bailey S (1994) The Ccp4 Suite - Programs for Protein Crystallography. Acta Crystallographica Section D-Biological Crystallography 50: 760–763.
- 37. Winn MD, Isupov MN, Murshudov GN (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallographica Section D-Biological Crystallography 57: 122–133.
- 38. Winn MD, Murshudov GN, Papiz MZ (2003) Macromolecular TLS refinement in REFMAC at moderate resolutions. Macromolecular Crystallography, Pt D 374: 300–321.