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Norman Sims
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Supplemental Online Material Supplemental Figures Supplemental Figure 1. A) Crosslinking efficiencies of 5 -biotin tagged oligonucleotides screened with the FASTDXL method 1.

1 Supplemental Online Material Supplemental Figures Supplemental Figure 1. A) Crosslinking efficiencies of 5 -biotin tagged oligonucleotides screened with the FASTDXL method 1. Reactive and nonreactive cysteine positions are shown as large magenta or small yellow spheres, respectively. The sequence of the archetypal screening oligonucleotide is shown above the protein in register with the nucleic acid as it appears in the structure. Individually modified nucleic acid crosslinking positions are shown in orange. The portion of the FASTDXL screening 1 / 10

2 oligonucleotide translated into crystal trials is capitalized. B) Initial 2mF o -df c (1σ, blue) and mf o -df c (2.5σ, green) maps of the Asn232Cys complex. Maps were calculated after one round of positional and ADP refinement with Refmac 3, using an apo model of the RPD (purple) as a starting point. Superposition of the refined Arg320Cys/ssDNA complex (yellow) upon the Asn232Cys model illustrates that the template binding groove accommodates a similar conformation of nucleic acid within both crystal forms. Severely anisotropic diffraction precluded the generation of a fully refined model, but attempts to model this density with nucleic acid in the opposite orientation (3-5 towards the active site) were unsuccessful. 2 / 10

3 Supplemental Figure 2. 1 H- 15 N HSQC spectrum of the apo wildtype E. coli DnaG RNA Polymerase Domain colored per Figure 3 of the main text. 3 / 10

4 Supplemental Figure 3. 1 H- 15 N HSQC spectrum of the wildtype E. coli DnaG RNA Polymerase Domain with 300 µm ssdna colored per Figure 3 of the main text. 4 / 10

5 Supplemental Figure 4. 1 H- 15 N HSQC spectrum of the wildtype E. coli DnaG RNA Polymerase Domain with 600 µm ssdna colored per Figure 3 of the main text. 5 / 10

6 Supplemental Figure 5. Establishment of statistical equilibrium in alignments and criterion for selection of perturbations 4. A) The mean ΔE stat between five unconserved sites (alignment positions 170, 173, 195, 198, and 400) in a nonredundant multiple sequence alignment of 210 bacterial primases is relatively insensitive to the elimination of a large fraction of the sequences. B) The ΔΔE stat between the same five unconserved sites exhibits low coupling energies with even relatively small subalignments. The minimal subalignment was chosen to be 80 sequences, based on a threshold of kt. 6 / 10

7 Supplemental Methods FASTDXL screening for stable protein-dna complexes. We have recently reported details of this methods elsewhere 1. Briefly, 5 -biotin-tagged oligonucleotides containing a single O6-phenyl-dI modification were purchased from Operon and derivatized by reaction with 1 M cystamine for 18 hours at 68 C. Cysteine point mutations were introduced into the E. coli DnaG RNA Polymerase Domain (residues ) using the QuikChange method. Stable protein-dna complexes were identified by mixing pools of cysteine mutants with cystamine-derivatized oligonucleotides. Successfully crosslinked mutant/oligonucleotide combinations were identified by MALDI/TOF and MS/MS. Several different DnaG/ssDNA combinations crosslinked particularly efficiently (Supplemental Fig. 1A) and were individually scaled up and moved to crystal trials 1. Preparation of protein-dna complexes. The E. coli DnaG RNA Polymerase Domain was cloned into the psv271 vector 2 and expressed as an N-terminal TEV-cleavable His 6 fusion in BL21(DE3) codon+ cells by inducing for 8 hours with 0.5 mm IPTG at a cell density of 0.5 at 600 nm. Following sonication and centrifugation, the crude cell lysate was passed over a Hi-Trap MC-Ni column (Amersham) in Buffer A (0.5 M NaCl, 20 mm HEPES, 10% glycerol (v/v)) with 10 mm imidazole, and bound protein eluted in a single step with Buffer A M imidazole. The His 6 tag was removed by overnight incubation at 4 C with His-tagged TEV protease, and further impurities removed by passing the solution over a second nickel column and collecting the flowthrough. A Sephacryl S-200 gel filtration column (Amersham) equilibrated in Buffer A was used as a 7 / 10

8 final step to remove aggregated protein. The protein/dna complex was formed by mixing each polymerase domain mutant with the thiolated nucleic acid at a 2:1 molar ratio as described 1, and allowing them to crosslink for 48 hours at 4 C. Stable complexes were separated from free protein using a Hi Trap Q column (Amersham) in a gradient of Buffer A from mm NaCl, and free nucleic acid was removed with a final S-200 gel filtration column. 8 / 10

9 Supplemental Movie 1. Interpolated ssdna tracking via the basic groove. The protein cores of each complex in the asymmetric unit of the Arg320Cys crystal form were superposed and linear interpolation was performed over thirty steps 5,6. 9 / 10

10 References 1. Corn, J.E. & Berger, J.M. FASTDXL: a generalized screen to trap disulfidestabilized complexes for use in structural studies. Structure 15, (2007). 2. Corn, J.E., Pease, P.J., Hura, G.L. & Berger, J.M. Crosstalk between primase subunits can act to regulate primer synthesis in trans. Mol Cell 20, (2005). 3. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Cryst D 53, (1997). 4. Shulman, A.I., Larson, C., Mangelsdorf, D.J. & Ranganathan, R. Structural determinants of allosteric ligand activation in RXR heterodimers. Cell 116, (2004). 5. Flores, S. et al. The Database of Macromolecular Motions: new features added at the decade mark. Nucleic Acids Res 34, D (2006). 6. Krebs, W.G. & Gerstein, M. The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework. Nucleic Acids Res 28, (2000). 10 / 10

nature methods Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates

nature methods Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates Audur Magnusdottir, Ida Johansson, Lars-Göran Dahlgren, Pär Nordlund & Helena Berglund Supplementary