Supplemental Material for

Structure of an Ebf1-DNA complex reveals unusual DNA

recognition and structural homology with Rel proteins

Nora Treiber, Thomas Treiber, Georg Zocher and Rudolf Grosschedl

Inventory:

Supplemental figures 1 - 8 with figure legends

Supplemental methods

Supplemental tables 1 - 3

Supplemental references

Supplemental Figure S1: Structure model of the additional non-DNA-bound Ebf1 monomer in crystal form II. In addition to the DNA-bound dimer the Ebf1-DBD crystal form II contains an additional protein monomer. The presented structure model shows that the core structure

consisting of the -sandwich is unaltered in comparison to the DNA-bound domains. In contrast, the DNA-contacting loops are unstructured in this monomer and the Zn-knuckle has lost the central ion and is involved in a crystal contact. For stretches that diverge from the conformation in the DNA-bound monomers or are not structured, the corresponding regions in the DNA-bound structures are shown as dashed lines. Colouring and labelling of secondary structure elements are as in Fig. 1a.

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Supplemental Figure S2: Structure of the Zinc-knuckle a) Cartoon of the zinc-knuckle structure from the model of the Ebf1-DBD bound to DNA. b) Schematic drawing of the zinc-knuckle showing the sequence and secondary

structure elements (blue – helix, green – -sheet). DNA-binding residues are marked red. The coordination of the central ion is shown as black lines.

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Supplemental Figure S3: Secondary structure elements of the Ebf1 DNA-binding domain

The secondary structure elements are annotated as arrows (-sheets) and cylinders

(helices) underneath the amino acid sequence. As in Figure 1, the -strands of the sandwich structure are shaded in yellow and orange and labelled beginning with A. Strands outside the sandwich structure are shown in green and labelled X-Z. Helices are blue and numbered. 310 helices are shown as light grey cylinders. Amino acids that form hydrogen bonds to DNA bases are highlighted in green (central binding module), blue (GH loop) and red (zinc knuckle). The four residues that coordinate the Zn2+-ion in the zinc-knuckle are highlighted in yellow.

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Supplemental Figure S4: Structure based alignment of the TIG-domains of Ebf1 and Rel-family factors

The amino acid sequences of TIG-domains of Ebf1 and seven Rel-family transcription factors are shown. A structure based alignment

was created using the following entries from the PDB database: NF-B p50 – 1SVC, NF-B p65 – 2RAM, NF-B p52 – 1A3Q, Rel-B –

2V2T, C-Rel – 1GJI, NFAT5 –1IMH, NFAT1 – 1P7H. The numbers of the first and last residue of the TIG domain are given. Residues

that occupy structurally equivalent positions to an Ebf1 residue are shown in upper case and are aligned, residues that do not have an

equivalent in the TIG-domain of Ebf1 are shown in lower case. The secondary structure of the Ebf1 TIG-domain is annotated in the

topmost line. Residues that participate in the formation of the dimer interface are underlined, residues that form hydrogen bonds or

salt bridges in the interfaces are written in red. For NFAT1, which forms asymmetric dimer contacts in the reported crystal structure,

residues from the two interfaces are shown in red and blue. Sequence conservation is highlighted in shades of yellow, fully conserved

positions are marked with an asterisk.

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Supplemental Figure S5: Structure based alignment of the DBD of Ebf1 with Ig-fold transcription factors The DNA-binding domain of Ebf1 was superposed with the Ig-fold DBDs of

structurally homologous transcription factors identified by DALI (Table S3). Structure

files used are: NFAT1 – 1P7H, NFAT – 1IMH, STAT – 1UUS, NF-kB P65 – 2RAM,

c-Rel – 1GJI, p53 – 1HU8, Runx1 – 1EAO. Contiguous stretches of structurally

equivalent secondary structure are shown aligned to the Ebf1 sequence. The

secondary structure of Ebf1 is shown above the alignment. Amino acid residues that

are identical to Ebf1 are shaded orange, similar residues are shaded yellow. A

vertical line behind a residue indicates a not aligned insertion at this position.

Sequence numbers of the aligned segments are given behind each line of sequence.

Notably, the order of the aligned segments does not follow the primary sequence of

the aligned factors due to the unique topology of the DBD of Ebf1.

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b

c

a

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Supplemental Figure S6 (previous page): Stereoscopic view of the three DNA-binding modules of Ebf1 The DNA contacts of the GH-loop (a), the central module (b) and the zinc-knuckle (c) are shown. Residues that form hydrogen bonds to the DNA are depicted as ball-and-stick model, the hydrogen bonds are indicated as dashed lines (black – backbone contact, pink – base contact, blue – protein-protein contacts). Nucleotides and amino acid residues involved in the contacts are labelled. The DNA bases of the conserved Ebf1-recognition motif are colour coded as in Figure 3.

Supplemental Figure S7: Closeup view of the DBD-dimerization contact The main contact interface of the Ebf1 DNA-binding domains is depicted as viewed from underneath the DNA-bound complex. The sidechains of the residues 146-149, which constitute the main part of the interface, are shown as ball-and-stick model. K146 and N147 contribute the largest contact areas and were mutated to disrupt dimerization. These residues are highlighted in orange and labelled.

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Supplemental Figure S8: Expression controls of mutant Ebf1 proteins used in the validation experiments a) Ebf1 protein for the gel-shift assays was produced by coupled in vitro transcription and translation using a reticulocyte lysate system (Promega). In a parallel set of samples 35S-labelled methionine was incorporated into the proteins and the expression level of the individual mutants was assessed by SDS-PAGE followed by autoradiography. The positions of Ebf1 and unincorporated 35S-methionine are indicated. b) Immunoblot analysis of Ebf1-expression in retrovirally transduced BAF/3 cells. Levels of the housekeeping gene Gapdh are shown as loading control.

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SUPPLEMENTAL METHODS

Protein expression, purification and crystallization

Ebf126-422 without a stop codon was cloned into the pET23d vector (Novagen)

resulting in a C-terminally His6-tagged construct for inducible expression in E.coli.

Expression was performed in the E.coli strain Rosetta (Novagen) for 20 hours at

18°C after induction with 1 mM IPTG at OD600=0.7. The bacteria were lysed in His-

buffer A (300 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 10% (v/v)

Glycerol, 40 mM Imidazole pH 7.4). The filtered lysate was applied to a Ni2+-loaded

IMAC column (Biorad) and Ebf126-422 was eluted by an imidazole gradient. The peak

fractions, which also showed a marked precipitation of protein, were pooled and

supplemented with twenty percent of their volume of glycerol and 10 mM final

concentration of DTT. This solution was warmed to 25-30°C in a water bath and then

stirred gently at room temperature until the protein precipitate had dissolved. For a

second purification step the protein solution was applied to a heparin-sepharose

column (GE Healthcare) using Heparin buffer A (20 mM HEPES pH 7.4, 400 mM

NaCl, 10% (v/v) Glycerol, 10 mM DTT) as loading buffer. Pure Ebf126-422 was eluted

with a salt gradient (400-1000 mM). The eluate was concentrated to roughly 20 mg

Ebf1 per milliliter. Double stranded oligonucleotide was prepared by annealing the

palindromic single strand in 50 mM HEPES pH 7.4, 200 mM NaCl, 2 mM MgCl2. For

the assembly of Ebf1:DNA complexes the protein was incubated with a slight molar

excess (1:1.2) of the oligonucleotide and then subjected to size exclusion

chromatography in GPC buffer (20 mM HEPES pH 7.4, 200 mM NaCl, 10 mM DTT).

The complex was concentrated to 8-10 mg/ml for crystallization. Crystals of the

Ebf26-422-complex grew at 37°C in 10% (w/v) PEG 4000, 100 mM NaCitrate pH 5.4

and 10% (v/v) isopropanol within one day after microseeding. For cryo protection,

crystals were briefly soaked in the crystallization buffer with 10% (v/v) PEG 400 and

then flash-frozen. The reproducibility of this crystal form was poor and most crystals

were not suitable for diffraction experiments. In fact only a single crystal yielded a

clear diffraction pattern with an acceptable resolution, making phasing by heavy

metal soaks or with crystals of selenomethionine-substituted protein impossible.

For purification of the isolated DNA-binding domain of Ebf1, the TIG-domain was

deleted from the expression construct and replaced with a TEV protease cleavage

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site. After the initial Ni2+-IMAC purification, the protein was dialyzed over night

against His-buffer A in the presence of 1 mg TEV protease per 100 mg of Ebf1. The

cleaved protein was again passed over the Ni2+-IMAC column to remove uncleaved

protein as well as the His-tagged HLH-domain, leaving the almost pure Ebf1-DBD in

the flowthrough. The protein was precipitated by addition of 50% saturation (295 g/l)

of ammonium sulfate and redissolved in 5 ml of GPC buffer. For final polishing a size

exclusion chromatography on Superdex 200 material was performed. The protein

was concentrated to 15 mg/ml and cocrystallized with 4.3 mg/ml of double stranded

oligonucleotide. Crystals of form I grew within a few days at 20°C in 6%

(w/v) PEG 4000, 100 mM MES pH 5.7 and 200 mM KCl, while crystal form II was

found after one week in 10% (w/v) PEG 4000 and 200 mM KCl. Crystal form I

crystals were cryo-protected by soaking briefly in crystallization buffer with 20% (v/v)

glycerol before flash-freezing. For crystal form II, 100% (v/v) glycerol was added

directly to the drop and crystals were immediatly flash-frozen.

Solutions of Ebf1 were generally handled at room temperature as low temperatures

lead to increased precipitation (which can be reverted by heating the solutions to

37°C).

Structure determination and quality of the models

The structure of the DBD was determined using phases from a Ta6Br14-soaked

crystal (crystal form I). Initial phases were yielded using SHARP(de la Fortelle and

Bricogne 1997) and subsequent density modification as implemented in SHARP.

The resulting electron density allowed for building an initial model including the DNA

duplex using COOT(Emsley and Cowtan 2004). This model was subsequently used

as a search model for phasing the data derived from crystal form II by molecular

replacement with PHASER(McCoy et al. 2007). Refinement was carried out using

REFMAC5.0(Murshudov et al. 1997) of the CCP4-suite and manual model building in

COOT. Later on, TLS-refinement was implemented using the single domains as

TLS-groups(Winn et al. 2001). Water was built using COOT. The final model of the

DNA-bound Ebf1 dimer contains residues 35-239 and the TEV-cleavage site

(ENLYFQ) as well as the complete DNA duplex.

The refined model of the DBD-dimer bound to DNA was used as a search model for

molecular replacement of the native data of crystal form I. To remove model bias, a

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simulated annealing was performed using PHENIX(Adams et al. 2010). The model

was subsequently refined as described above.

The data for EBF26-422 was also phased by molecular replacement using the refined

high-resolution model of the DBD-dimer (aa 53-239) bound to DNA as a search

model. Two complexes could be placed into the asymmetric unit with clear, but

incomplete density emerging for the TIG-domains. Numerous subsequent building

and refinement cycles using COOT and REFMAC5.0, respectively, steadily improved

the electron density until four TIG-domains could be build with high confidence. In

each EBF-dimer, only one TIG-domain is linked by visible electron density to the

DBD-domain (chain A and E), while the second linker is missing. Furthermore, the

N-termini including the first helix assume a slightly different conformation in the

different monomers, which is probably due to crystal packing effects. Refinement of

the IPT-domains further improved phases, resulting in additional electron density at

the C-termini of one complex. This density could clearly be interpreted as a four helix

bundle, with more density for one linker and parts of the loop regions emerging

during refinement. However, the electron density for side chains was very weak. As

MyoD shows a clear sequence similarity with the first two helices of the HLH-domain

of Ebf1, we superposed a published structure of a MyoD dimer(Ma et al. 1994) onto

the helices which revealed an almost identical conformation of the helix bundle. The

homology in combination with the length of the visible linker was used to dock the

sequence of the Ebf1 HLH-domain. As Rfree did not improve upon inclusion of these

side chains into the Ebf1-structure, the proposed structure of the HLH-domain retains

a model-like character.

The geometry of the refined structures was analysed with MOLPROBITY(Davis et al.

2007) and found to be excellent (apart from the HLH domain).

Validation experiments. Electrophoretic mobility shift assays were performed as

described (Hagman et al. 1995). Wild type and mutant Ebf1 proteins were generated

by in vitro transcription and translation using the TNT T7 quick coupled

Transcription/Translation system (Promega) and mutants of Ebf1 in the pET23d

vector (Novagen). Per gel shift 10µl of the translation reaction were used. For

activation of the endogenous Igll1 gene, BAF/3 cells were retrovirally transduced with

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Ebf1-IRES-GFP as described (Treiber et al. 2010), and sorted for GFP expression

24h after transduction. RNA and protein of the sorted cells were isolated using trizol

reagent (Invitrogen) and the RNA was reverse transcribed with Superscript II reverse

transcriptase and random hexamer primers. The expression levels of the Igll1 and

Tpi genes were probed by quantitative PCR using specific primers.

Supplemental Table S1: Data collection statistics

DBD crystal form I EBF 26-422

nativ Ta-labeled

DBD crystal form II

peak inflection high remote

beamline/wavelength PXIII / 1.0000 PXIII / 1.2551 PXIII / 1.2556 PXIII / 1.2425 PXIII / 1.0097 BLX14.2 / 0.9537

space group P6522 P21 P1

unit cell parameters a, b, c , β, γ

70.8, 70.8, 601.6

70.9, 70.9, 605.4

71.3, 100.8, 72 90, 101.5, 90

70.5, 78.7, 103.6 89.0, 90.0, 77.8

solvent [%] 65.0 65.3 57.8 60.9

resolution [Å] 30 - 3.0 (3.1 - 3.0) 30 - 3.1 (3.3 - 3.1) 29.3 - 2.4 (2.5 - 2.4) 50 - 2.8 (3.0 - 2.8)

unique reflections 19172 (1567) 30882 (5742) 30600 (5730) 30640 (5727) 38953 (4366) 52163 (9689)

completeness [%] 98.9 (89.9) 99.9 (100) 99.2 (99.8) 99.1 (99.7) 99.6 (98.0) 96.7 (96.2)

multiplicity 13.1 (9.9) 13.9 (13.3) 5.7 (5.4) 5.7 (5.4) 4.6 (4.4) 2.3 (2.2)

Rsym-I [%] 18.2 (59.1) 12.1 (44.5) 11.2 (38.5) 10.9 (36.5) 8.3 (45.9) 8.9 (46.0)

average I/σ I 11.8 (2.6) 20.5 (5.6) 12.9 (4.0) 13.2 (4.2) 14.8 (3.2) 9.9 (2.3)

Wilson B-factor [Å2] 41.7 50.6 49.6 49.0 39.0 49.2

protomers/asu protein monomers dsDNA

2 1

3 1

4 2

* numbers in paratheses refer to the last resolution shell

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Supplemental Table S2: Refinement statistics

DBD crystal form I

DBD crystal form II

EBF 26-422

resolution range 30 - 3.0 29.3 - 2.4 50 - 2.8

no. reflections 19172 38953 52162

no. atoms polypeptide DNA Zn citrate

3413 896

2 -

5004 896

2 -

9954 1792

4 65

no. water molecules 39 445 150

structured polypeptide A B E F

35 - 246

35 - 247

-

-

35 - 240

35 - 247

27 - 93/106 - 133/

150 - 162/175 - 200/ 205 - 221/223 - 235

-

36 - 338/342 - 355/

367 - 386 36 - 242/252 - 357/

365 - 383 36 - 338

36 - 241/250 - 341

average B-factors [Å2] protein DNA solvent

57.8/66.0 58.5/57.4

49.3

35.8/36.0/36.2

36.9/35.5 38.7

56.7/56.9/49.6/45.9 39.8/41.7/37.8/37.4

37.4

Rcryst [%] 22.2 17.1 20.4

Rfree [%] (5% test set) 28.1 21.9 26.4

rmsd from ideal Bond lengths [Å] Bond angles [°]

0.009 1.395

0.010 1.350

0.010 1.411

Ramachandran angles favoured regions [%] allowed regions [%]

96.4 3.6

97.7 2.3

95.5 4.5

TLS-groups protein DNA

A 35 - 246 B 35 - 247

C 1 - 22 D 1 - 22

A 35 - 240 B 35 - 247 E 27 - 235

C+D 1 - 22

A,B,E 36 - 242, F 36 - 241

A,E 243 - 338 B 252 - 338, F 250 - 341 A 342 - 355 + 367 - 386 B 339 - 357 + 365 - 383

C+D 1 - 22 G+H 1 - 22

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Supplemental Table 3: Structural homologs of the Ebf1 DNA-binding domain according to DALI(Holm et al. 2008)

Z-Score PDB-code* Protein name Organism C-r.m.s.d. No. of aligned residues

Reference

6.7/ 6.6 1PZU/1P7H NFAT1 Homo sapiens 3.1/ 3.3 108/ 111 (Giffin et al. 2003; Jin et al. 2003)

6.4 1IMH TonEBP/NFAT5 Homo sapiens 3.6 110 (Stroud et al. 2002)

6.4 1UUS STAT Dictyostelium discoideum 3.3 109 (Soler-Lopez et al. 2004)

6.3 1CWV invasin Yersinia pseudotuberculosis 3.3 98 (Hamburger et al. 1999)

6.3 2RAM NF-kB p65 homodimer Mus musculus 3.7 99 (Chen et al. 1998)

6.2 1GJI c-Rel Gallus gallus 3.7 102 (Huang et al. 2001)

6.2 1QFH gelation factor Dictyostelium discoideum 3.3 101 (McCoy et al. 1999)

6.1 1HU8 p53 Mus musculus 3.4 114 (Zhao et al. 2001; Cho et al., 1994)

6.0 1EAO Runx1 Mus musculus 2.8 92 (Backstrom et al. 2002)

5.8 1A3Q NF-kB p52 Homo sapiens 3.9 100 (Cramer et al. 1997)

5.8 2H1L large T-antigen Homo sapiens 3.4 114 (Lilyestrom et al. 2006)

5.7 2DS4 tripartite motif protein 45 Homo sapiens 3.3 102 unpublished

5.6 3G6J complement C3b b-chain Homo sapiens 3.9 94 (Katschke et al. 2009)

5.6 3H7L endoglucanase Vibrio parahaemolyticus 2.6 83 unpublished

5.5 3DO7 Rel-B Mus musculus 3.6 100 unpublished

5.5 2J3S filamin-A Homo sapiens 3.0 87 (Lad et al. 2007)

5.5 2A74 complement component C3c

Homo sapiens 3.6 96 (Janssen et al. 2005)

5.3 1BVO gambif1 Anopheles gambiae 3.4 95 (Cramer 1999)

5.1 1SVC NF-kB p50 Homo sapiens 2.9 87 (Muller et al. 1995)

*For each protein only the PDB-entry with the highest Z-score is cited.

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Supplemental references Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung,

L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J., Richardson, D.C., Richardson, J.S., Terwilliger, T.C., and Zwart, P.H. 2010. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66: 213-221.

Backstrom, S., Wolf-Watz, M., Grundstrom, C., Hard, T., Grundstrom, T., and Sauer, U.H. 2002. The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding. J Mol Biol 322(2): 259-272.

Chen, Y.Q., Ghosh, S., and Ghosh, G. 1998. A novel DNA recognition mode by the NF-kappa B p65 homodimer. Nat Struct Biol 5(1): 67-73.

Cho Y, Gorina S, Jeffrey PD, Pavletich NP. 1994. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265: 346-355.

Cramer, P., Larson, C.J., Verdine, G.L., and Muller, C.W. 1997. Structure of the human NF-kappaB p52 homodimer-DNA complex at 2.1 A resolution. Embo J 16(23): 7078-7090.

Cramer, P., Varrot, A., Barillas-Mury, C., Kafatos, F.C., and Muller, C.W. 1999. Structure of the specificity domain of the Dorsal homologue Gambif1 bound to DNA. Structure 7(7): 841-852.

Davis, I.W., Leaver-Fay, A., Chen, V.B., Block, J.N., Kapral, G.J., Wang, X., Murray, L.W., Arendall, W.B., 3rd, Snoeyink, J., Richardson, J.S., and Richardson, D.C. 2007. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35(Web Server issue): W375-383.

de la Fortelle, E. and Bricogne, G. 1997. Maximum-Likelihood Heavy-Atom Parameter Refinement for the Multiple Isomorphous Replacement and Multiwavelength Anomalous Diffraction Methods. Methods in Enzymology 276: 472-494.

Emsley, P. and Cowtan, K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1): 2126-2132.

Giffin, M.J., Stroud, J.C., Bates, D.L., von Koenig, K.D., Hardin, J., and Chen, L. 2003. Structure of NFAT1 bound as a dimer to the HIV-1 LTR kappa B element. Nat Struct Biol 10(10): 800-806.

Hamburger, Z.A., Brown, M.S., Isberg, R.R., and Bjorkman, P.J. 1999. Crystal structure of invasin: a bacterial integrin-binding protein. Science 286(5438): 291-295.

Holm, L., Kaariainen, S., Rosenstrom, P., and Schenkel, A. 2008. Searching protein structure databases with DaliLite v.3. Bioinformatics 24(23): 2780-2781.

Huang, D.B., Chen, Y.Q., Ruetsche, M., Phelps, C.B., and Ghosh, G. 2001. X-ray crystal structure of proto-oncogene product c-Rel bound to the CD28 response element of IL-2. Structure 9(8): 669-678.

Janssen, B.J., Huizinga, E.G., Raaijmakers, H.C., Roos, A., Daha, M.R., Nilsson-Ekdahl, K., Nilsson, B., and Gros, P. 2005. Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437(7058): 505-511.

Jin, L., Sliz, P., Chen, L., Macian, F., Rao, A., Hogan, P.G., and Harrison, S.C. 2003. An asymmetric NFAT1 dimer on a pseudo-palindromic kappa B-like DNA site. Nat Struct Biol 10(10): 807-811.

Katschke, K.J., Jr., Stawicki, S., Yin, J., Steffek, M., Xi, H., Sturgeon, L., Hass, P.E., Loyet, K.M., Deforge, L., Wu, Y., van Lookeren Campagne, M., and Wiesmann, C. 2009. Structural and functional analysis of a C3b-specific antibody that selectively inhibits the alternative pathway of complement. J Biol Chem 284(16): 10473-10479.

Lad, Y., Kiema, T., Jiang, P., Pentikainen, O.T., Coles, C.H., Campbell, I.D., Calderwood, D.A., and Ylanne, J. 2007. Structure of three tandem filamin domains reveals auto-inhibition of ligand binding. Embo J 26(17): 3993-4004.

Lilyestrom, W., Klein, M.G., Zhang, R., Joachimiak, A., and Chen, X.S. 2006. Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor. Genes Dev 20(17): 2373-2382.

Ma, P.C., Rould, M.A., Weintraub, H., and Pabo, C.O. 1994. Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation. Cell 77(3): 451-459.

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McCoy, A.J., Fucini, P., Noegel, A.A., and Stewart, M. 1999. Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod. Nat Struct Biol 6(9): 836-841.

McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., and Read, R.J. 2007. Phaser crystallographic software. J Appl Crystallogr 40(Pt 4): 658-674.

Muller, C.W., Rey, F.A., Sodeoka, M., Verdine, G.L., and Harrison, S.C. 1995. Structure of the NF-kappa B p50 homodimer bound to DNA. Nature 373(6512): 311-317.

Murshudov, G.N., Vagin, A.A., and Dodson, E.J. 1997. Refinement of Macromolecular Structures by the Maximum-Likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240-255.

Soler-Lopez, M., Petosa, C., Fukuzawa, M., Ravelli, R., Williams, J.G., and Muller, C.W. 2004. Structure of an activated Dictyostelium STAT in its DNA-unbound form. Mol Cell 13(6): 791-804.

Stroud, J.C., Lopez-Rodriguez, C., Rao, A., and Chen, L. 2002. Structure of a TonEBP-DNA complex reveals DNA encircled by a transcription factor. Nat Struct Biol 9(2): 90-94.

Winn, M., Isupov, M., and Murshudov, G.N. 2001. Use of TLS parameters to model anisotropic displacement in macromolecular refinement. Acta Crystallogr D Biol Crystallogr 57: 122-133.

Zhao, K., Chai, X., Johnston, K., Clements, A., and Marmorstein, R. 2001. Crystal structure of the mouse p53 core DNA-binding domain at 2.7 A resolution. J Biol Chem 276(15): 12120-12127.