The authors wish to thank the PXRR staff at the NSLS (Brookhaven National Laboratory) for assistance during data collection and Monica Pillon for help with DNA purification. This work was supported by the Canadian Institutes of Health Research (MOP 67189).

1. Protein Purification
The flexible linker connecting the N- (oligomerization) and C-terminal (DNA binding) domains of SeqA aids the recognition of hemimethylated GATC repeats separated by one to three turns on the DNA. For this study, we used a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) with a point mutation in the N-terminal domain that prevents further oligomerization and a shortened linker that restricts DNA binding to tandem GATC repeats separated exclusively by one turn on the DNA (Figure 1)2,8.
<ol>
 <li>Transform BL21 (DE3) cells with the plasmid encoding SeqA under the control of T7 promoter,</li>
 <li>Plate the transformation reaction in LB-agar plates including 100 &mu;g/ml ampicillin,</li>
 <li>Pick mixed colonies to inoculate a small-scale overnight culture (LB media with 100 &mu;g/ml ampicillin),</li>
 <li>The next morning inoculate a 1 L of media using a 1:100 dilution of the overnight culture,</li>
 <li>Grow the cells to an OD600 of ~0.7 and induce protein production by addition of isopropyl &beta;-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM,</li>
 <li>Continue the incubation for 3 hr at 37 &deg;C with orbital shaking and then harvest the cells by centrifugation (10 min at 3,300 g),</li>
 <li>Resuspend the cell pellet in purification buffer A and lyse by sonication,</li>
 <li>Clear the lysate by centrifugation (40 min at 39,000 g) and load the supernatant onto a heparin column equilibrated with purification buffer,</li>
 <li>Elute SeqA using a linear gradient to 1 M NaCl (SeqA elutes at ~0.7 M NaCl),</li>
 <li>Pool the SeqA-containing fractions together, dilute to lower the ionic strength of the sample and load into a cation-exchange chromatography column equilibrated with purification buffer,</li>
 <li>Using a linear salt gradient, pure SeqA elutes at ~0.4 M NaCl,</li>
 <li>Pool the SeqA-containing fractions together, concentrate (3 mg/ml) and store in storage buffer.</li>
</ol>
2. DNA Purification
<ol>
 <li>Order complementary unmethylated and methylated oligonucleotides from your favorite company,</li>
 <li>Dissolve 1 &mu;mol of each lyophilized single strand DNA in 800 &mu;l of autoclaved ddH2O, vortex and let it sit for 10-20 min,</li>
 <li>Add 800 &mu;l of preheated 2X loading buffer to each oligonucleotide,</li>
 <li>For 20-30 nucleotides long oligonucleotides, prepare a large 10% denaturing gel (160 x 250 x 1 mm): 
 * Mix well 80 ml of 10% PAGE mix, 80 &mu;l TEMED and 800 &mu;l ammonium persulfate per gel and pour, 
 * Once polymerized, remove the comb and rinse the wells with ddH2O thoroughly, 
 * Assemble the gels on gel cast including cooling plate and then fill the top and bottom reservoirs with running buffer (1X TBE), 
 * Pre-run the gel at 700-750 V to warm the gel up to 55 &deg;C, 
 * Stop the run and rinse the wells thoroughly with running buffer.</li>
 <li>Heat the oligonucleotides to 90 &deg;C for 2 min,</li>
 <li>Vortex and spin the samples and immediately before loading the gel,</li>
 <li>Run the gel at ~700 V and stop it once your oligonucleotide has migrated halfway. (Note that on a 10% polyacrylamide gel bromophenol blue co-migrates with oligonucleotides ~20 bases long and xylene cyanol FF with ~60 bases long),</li>
 <li>Stop the gel, disassemble it from the gel box and remove the spacers,</li>
 <li>On a flat surface, remove one glass plate and cover the gel with plastic wrap,</li>
 <li>Turn the gel around, remove the other glass plate and cover it with plastic wrap,</li>
 <li>Mark the bands using UV light and a fluorescent plate behind the gel to see the DNA shadow,</li>
 <li>Cut the band with a razor blade into small pieces and transfer them into a sterile 15 ml tube,</li>
 <li>Add 9 ml of elution buffer and elute overnight at 37 &deg;C with agitation,</li>
 <li>Carefully transfer the solution to an autoclaved centrifuge tube using a gel-loading pipette tip to avoid transferring acrylamide pieces and add 1 ml of 3 M sodium acetate at pH 7 (1:10 dilution) plus 25 ml of chilled 100% ethanol (2.5 volumes),</li>
 <li>Incubate at -20 &deg;C for at least 3 hr,</li>
 <li>Spin down and transfer the supernatant to a separate tube,</li>
 <li>Dry the pellet on the speed-vac at medium heat,</li>
 <li>Resuspend the pellet in 400 &mu;l of autoclaved ddH2O and transfer to a fresh tube,</li>
 <li>Add 40 &mu;l of 3 M sodium acetate at pH 7 and 1 ml of 100% ethanol; mix well (vortex) and incubate 30 min at room temperature followed by 30 min at -20 &deg;C,</li>
 <li>Spin for 15 min at 18,000 g and discard the supernatant,</li>
 <li>Rinse the pellet with 100 &mu;l of 70% cold ethanol to remove residual salt from the pellet and spin for 6 min at 18,000 g. Discard the ethanol and dry the pellet on speed-vac,</li>
 <li>Resuspend the pellet in a total of 100 &mu;l of autoclaved ddH2O. Measure the concentration of the oligonucleotide,</li>
 <li>To anneal the hemimethylated DNA duplexes, mix equimolar concentrations of the complementary single strands and heat the mixtures to 95 &deg;C in a water bath for 5 min and then let them cool down slowly to room temperature inside the water bath.</li>
</ol>
3. Protein-DNA Complex Formation and Analysis
<ol>
 <li>Mix equal volumes of purified SeqA&Delta;(41-59)-A25 (81 &mu;M) and hemimethylated DNA (81 &mu;M),</li>
 <li>Incubate at room temperature for 15 min and store at 4 &deg;C until you are ready to use it,</li>
 <li>Screen for crystallization conditions using commercial sparse-matrix screens,</li>
 <li>Once initial crystallization leads have been identified, optimize the conditions to grow diffraction quality crystals,</li>
 <li>Cryoprotect the resulting SeqA-DNA crystals by either increasing the amount of PEG 400 present in the crystallization solution to a final concentration of 25% (v/v) or adding 20% glycerol (v/v) to the crystallization solution,</li>
 <li>Scoop individual crystals with a nylon loop, and flash-freeze them in liquid nitrogen,</li>
 <li>Test the diffraction limit of each crystal at 100 K.</li>
</ol>
4. Representative Results
To obtain the crystal structure of SeqA&Delta;(41-59)-A25R bound to hemimethylated DNA, we consecutively optimized three parameters on the DNA: (i) separation between hemimethylated GATC sequences; (ii) length of the duplex; and (iii) the absence/presence of 5' overhangs.
Electro-mobility shift assays indicate that SeqA&Delta;(41-59)-A25R preferentially binds GATC repeats separated by 9-10 base pairs (Figure 1). Therefore, we initially screened duplexes 23-24 base pairs (bps) long containing two hemimethylated GATC sequences separated by either 9 or 10 bps. Three duplexes yielded nicely shaped crystals (Figure 2). Although none of the crystals diffracted to high resolution, the 23 bps long duplex with the two GATC sites separated by 9 bps diffracted X-rays better than the rest, indicating that a GATC separation of 9 bps was preferred for crystallization. Therefore, we fixed the inter-GATC spacing to 9 bps for all subsequent screens.
Generally, DNA crystallization is favored for duplex lengths corresponding to exact helical turns because multiple DNA molecules can stack head-to-tail to form a continuous B-DNA within the crystal9. Therefore, we shortened the overall length of the duplexes to 21 bps (i.e. two helical turns). While a 21 bps duplex with blunt ends did not yield diffraction quality crystals (data not shown), a 21 bps duplex with a single 5' overhang nucleotide on each end did yield crystals that diffracted to 5 &Aring; in our home source. The improvement on the diffraction limit suggested that end-to-end duplex association was indeed favoring crystal packing.
Since SeqA interacts with GATC sequences that are on the same face of the DNA, the opposite face of the DNA duplex should be exposed to the solvent. To further improve the crystals of the complex, we then modified the sequence of the duplex to include a CG dinucleotide between the two GATC sites to promote groove-backbone interactions with adjacent DNA molecules in the crystal through the opposite face of the duplex, a method that has been used to enhance DNA crystallization in the past10. However, the diffraction limit of the crystals grown with the CG-containing duplex was identical to those grown with a similar DNA duplex that did not contain the CG dinucleotide (Figure 3). This result indicated that groove-backbone interactions were not important in this case. We subsequently optimized the length of the overhangs by comparing the crystals grown with a DNA duplex that had two additional nucleotides at each 5' end. This change had a drastic effect on crystal morphology, as well as, diffraction limit indicating that the additional nucleotide dramatically changed the molecular contacts and enhanced crystal organization (Figure 3).
The crystal structure of SeqA&Delta;(41-59)-A25R bound to this last DNA duplex confirmed that the free face of the DNA duplex does not engage in crystal contacts, as expected from the limited effect of introducing a CG-dinucleotide. Despite the relative size of protein and DNA, most interactions between symmetry mates are mediated by protein-protein and protein-DNA interactions (Figure 4). Interestingly, in this particular case, the beneficial effect of a 5' dinucleotide overhang is not due to the formation of a pseudo-continuous DNA. Instead, the 5' end of the methylated strand projects away from the DNA axes and interacts with the proximal SeqA molecule of the complex, explaining why two nucleotides were strictly required to change the crystal packing8,11.
<img alt="Figure 1" src="/files/ftp_upload/4266/4266fig1.jpg" /> 
Figure 1. Binding of SeqA&Delta;(41-59)-A25R to hemimethylated DNA. (A) Schematic diagram of the domains of SeqA that mediate protein oligomerization and DNA binding. (B) Electrophoretic mobility shift assay of SeqA&Delta;(41-59)-A25R with DNAs containing two hemimethylated GATC sequences separated by an increasing number of base pairs (X). The left-most lane (labeled Blank) contains an equimolar mixture of DNAs with 5, 7, 12, 21, 25 and 34 base pairs between the two GATC sequences in the absence of SeqA&Delta;(41-59)-A25R. (C) Diagram depicting the three variables optimized on the DNA duplexes to obtain diffraction quality crystals.
<img alt="Figure 2" src="/files/ftp_upload/4266/4266fig2.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig2highres.jpg" /> 
Figure 2. Effect of varying the inter-GATC distance. Summary of the oligonucleotides used and crystals obtained with 23-24 bp long duplexes containing two hemimethylated GATC sites separated by 9 or 10 base pairs. All pictures of crystals were taken at the same magnification and the scale bar indicates 100 &mu;m. The resolution limit of each SeqA&Delta;(41-59)-A25R-DNA crystal is based on diffraction images collected on a Rigaku RU-300 X-ray generator system. <a href="https://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> 
Figure 3. Effect of varying the DNA ends. Summary of the oligonucleotides used and crystals obtained with 21 bps long duplexes containing two hemimethylated GATC sites separated by 9 base pairs and including 0, 1 or 2 nucleotide 5' end overhangs. All pictures of crystals were taken at the same magnification and the scale bar indicates 100 &mu;m. The resolution limit of each SeqA&Delta;(41-59)-A25R-DNA crystal is based on diffraction images collected at beamlines X12C and X29 (NSLS, BNL). <a href="https://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4266/4266fig4.jpg" /> 
Figure 4. Crystal packing of SeqA&Delta;(41-59)-A25R bound to DNA with dinucleotide overhang: Viewed from the (A) top and the side (B). The asymmetric unit contains two SeqA&Delta;(41-59)-A25R-DNA complexes where the protein is shown in grey while the DNA is shown in orange. Symmetry related SeqA&Delta;(41-59)-A25R-DNA molecules are shown in white for the protein and yellow for the DNA. This figure was prepared using PyMOL12. This figure is related to Movie 1. <a href="https://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
Movie 1. <a href="https://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank"> Click here to view movie</a>.

<ol>
	<li>Campbell, J. L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+oriC+and+the+dnaA+gene+promoter+are+sequestered+from+dam+methyltransferase+following+the+passage+of+the+chromosomal+replication+fork.">E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork.</a> Cell. 62, 967-979 (1990).</li><li>Guarn&#233;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+SeqA-N+filament:+implications+for+DNA+replication+and+chromosome+organization.">Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization.</a> Embo. J. 24, 1502-1511 (2005).</li><li>Brendler, T., Austin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+SeqA+protein+to+DNA+requires+interaction+between+two+or+more+complexes+bound+to+separate+hemimethylated+GATC+sequences.">Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.</a> Embo. J. 18, 2304-2310 (1999).</li><li>Jordan, S. R., Whitcombe, T. V., Berg, J. M., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+variation+in+DNA+length+yields+highly+ordered+repressor-operator+cocrystals.">Systematic variation in DNA length yields highly ordered repressor-operator cocrystals.</a> Science. 230, 1383-1385 (1985).</li><li>Tan, S., Hunziker, Y., Pellegrini, L., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+the+yeast+MATalpha2/MCM1/DNA+ternary+complex:+general+methods+and+principles+for+protein/DNA+cocrystallization.">Crystallization of the yeast MATalpha2/MCM1/DNA ternary complex: general methods and principles for protein/DNA cocrystallization.</a> J. Mol. Biol. 297, 947-959 (2000).</li><li>Rice, P. A., Yang, S., Mizuuchi, K., Nash, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+IHF-DNA+complex:+a+protein-induced+DNA+U-turn.">Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn.</a> Cell. 87, 1295-1306 (1996).</li><li>Yang, W., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+site-specific+recombinase+gamma+delta+resolvase+complexed+with+a+34+bp+cleavage+site.">Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site.</a> Cell. 82, 193-207 (1995).</li><li>Chung, Y. S., Brendler, T., Austin, S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+the+cooperative+binding+of+SeqA+to+a+tandem+GATC+repeat.">Structural insights into the cooperative binding of SeqA to a tandem GATC repeat.</a> Nucleic Acids Res. , (2009).</li><li>Anderson, J., Ptashne, M., Harrison, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocrystals+of+the+DNA-binding+domain+of+phage+434+repressor+and+a+synthetic+phage+434+operator.">Cocrystals of the DNA-binding domain of phage 434 repressor and a synthetic phage 434 operator.</a> Proc. Natl. Acad. Sci. U.S.A. 81, 1307-1311 (1984).</li><li>Timsit, Y., Moras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+self-fitting:+the+double+helix+directs+the+geometry+of+its+supramolecular+assembly.">DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.</a> Embo J. 13, 2737-2746 (1994).</li><li>Chung, Y. S., Guarne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+analysis+of+SeqA+bound+to+a+pair+of+hemimethylated+GATC+sites.">Crystallization and preliminary X-ray diffraction analysis of SeqA bound to a pair of hemimethylated GATC sites.</a> Acta Crystallogr Sect F Struct Biol Cryst Commun. 64, 567-571 (2008).</li><li>DeLano, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphic Systems. , (2002).</li></ol>

No conflicts of interest declared.

One of the biggest challenges in macromolecular X-ray crystallography is obtaining diffraction quality crystals. In the case of protein or protein-DNA complexes, this challenge is exacerbated due to the additional variables that must be optimized. It is widely believed that the length of the DNA and the presence of sticky overhangs to enhance association of neighboring DNA molecules in a longer pseudo-duplex are the main parameters to optimize. However, we have shown that the nature and length of these overhangs can have...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue #</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>TRIS</td>
 <td>Bioshop</td>
 <td>TRS003.5</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Fisher Scientific</td>
 <td>E478-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiotheitrol (DTT)</td>
 <td>Bio Basic Inc.</td>
 <td>DB0058</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Bioshop</td>
 <td>SOD002.10</td>
 <td></td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Caledon</td>
 <td>5350-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma-Aldrich</td>
 <td>S5016-500G</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate (SDS)</td>
 <td>Bioshop</td>
 <td>SDS001.500</td>
 <td></td>
 </tr>
 <tr>
 <td>Urea</td>
 <td>Bioshop</td>
 <td>URE001.5</td>
 <td></td>
 </tr>
 <tr>
 <td>40% 29:1 Bis/acrylamide</td>
 <td>Bio Basic Inc.</td>
 <td>A0007-500ml</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Boric acid</td>
 <td>EMD</td>
 <td>BX0865-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Xylene cyanol FF</td>
 <td>Bio-Rad</td>
 <td>161-0423</td>
 <td></td>
 </tr>
 <tr>
 <td>Bromophenol Blue</td>
 <td>Bioshop</td>
 <td>BR0222</td>
 <td></td>
 </tr>
 <tr>
 <td>Dual Adjustable Vertical Gel System</td>
 <td>C.B.C. Scientific Company Inc.</td>
 <td>DASG-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Index crystallization screen</td>
 <td>Hampton Research</td>
 <td>HR2-144</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Wizard I crystallization screen</td>
 <td>Emerald BioSystems</td>
 <td>EBS-WIZ-1</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Wizard II crystallization screen</td>
 <td>Emerald BioSystems</td>
 <td>EBS-WIZ-2</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Classics crystallization screen</td>
 <td>Qiagen</td>
 <td>130701</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Intelliplate trays </td>
 <td>Art Robbins Instruments</td>
 <td>102-0001-00</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions
 Protein purification buffer: 100 mM TRIS pH 8, 2 mM EDTA, 2 mM DTT and 5% glycerol.
 Protein storage buffer: 20 mM TRIS pH 8, 150 mM NaCl, 5 mM DTT, 0.5 mM EDTA and 5% glycerol.
 Gel loading mix: Add 20 g of sucrose, 25 mg of bromophenol blue, 25 mg of xylene cyanol FF, 1 ml of 10% w/v SDS and 10 ml of 10X TBE to 70 ml of autoclaved ddH2O. Stir with mild heating until sucrose is dissolved and adjust the final volume to 100 ml with autoclaved ddH2O. Store at 4 &deg;C.
 2X loading buffer: Add 11 g of urea to 10 ml of gel loading mix. Stir on a hot plate until urea dissolves. Aliquot in 2 ml tubes and store at 4 &deg;C. 
 10X PAGE mix: Mix 420.4 g of urea, 100 ml of 10X TBE (autoclaved), 250 ml of 40% 29:1 Bis/Acrylamide in ddH2O. Stir until totally dissolved and adjust volume to 1 liter. Store in dark bottles at 4 &deg;C.
 10X TBE: Dissolve 108 g of TRIS, 55 g of boric acid and 9.3 g of EDTA in 1 liter of ddH2O. Autoclave and store at room temperature.
 Elution buffer: Dilute 8 ml of 5 M NaCl, 2 ml of 1 M TRIS pH 7.5, 0.4 ml of 0.5 M EDTA pH 8 on 200 ml of ddH2O. Autoclave and store at room temperature.</td>
 </tr>
 </tbody>
 </table>

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

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9.9K Views.  McMaster University. Crystal structure of protein–DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Video: Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...