We thank the staff at the beamline X06DA at the Swiss Light Source, Paul-Scherrer-Institute, Villigen, Switzerland for support during synchrotron data collection. We are grateful to Monika Gompert for excellent technical support. The project was supported by the University of Cologne and grant INST 216/682-1 FUGG from the German Research Council. A PhD fellowship from the International Graduate School in Development Health and Disease to C.P. is acknowledged. The research leading to these results has received funding from the European Community&#39;s Seventh Framework Program (FP7/2007-2013) under grant agreement No. 283570 (BioStruct-X).

<ol>
	<li>Bouza, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Clostridium+difficile+infection:+understanding+the+healthcare+burden.">Consequences of Clostridium difficile infection: understanding the healthcare burden.</a> Clin Microbiol Infect. 18 (Suppl 6), 5-12 (2012).</li><li>O'Connor, J. R., Johnson, S., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection+caused+by+the+epidemic+BI/NAP1/027+strain.">Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain.</a> Gastroenterology. 136 (6), 1913-1924 (2009).</li><li>Mitchell, B. G., Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+and+Clostridium+difficile+infection:+a+review.">Mortality and Clostridium difficile infection: a review.</a> Antimicrob Resist Infect Control. 1 (1), (2012).</li><li>George, W. L., Sutter, V. L., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+agent-induced+diarrhea--a+bacterial+disease.">Antimicrobial agent-induced diarrhea--a bacterial disease.</a> J Infect Dis. 136 (6), 822-828 (1977).</li><li>George, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Clostridium+difficile+as+a+cause+of+pseudomembranous+colitis.">Identification of Clostridium difficile as a cause of pseudomembranous colitis.</a> Br Med J. 1 (6114), 695 (1978).</li><li>Bartlett, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrative+review:+the+new+epidemic+of+Clostridium+difficile-associated+enteric+disease.">Narrative review: the new epidemic of Clostridium difficile-associated enteric disease.</a> Ann Intern Med. 145 (10), 758-764 (2006).</li><li>Kelly, C. P., LaMont, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile--more+difficult+than+ever.">Clostridium difficile--more difficult than ever.</a> N Engl J Med. 359 (18), 1932-1940 (2008).</li><li>&#220;nal, C. M., Steinert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapeutic+strategies+for+Clostridium+difficile+infections.">Novel therapeutic strategies for Clostridium difficile infections.</a> Expert Opin Ther Targets. 20 (3), 269-285 (2016).</li><li>Kelly, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Monoclonal+Antibody,+Bezlotoxumab+Targeting+C.+difficile+Toxin+B+Shows+Efficacy+in+Preventing+Recurrent+C.+difficile+Infection+(CDI)+in+Patients+at+High+Risk+of+Recurrence+or+of+CDI-Related+Adverse+Outcomes.">The Monoclonal Antibody, Bezlotoxumab Targeting C. difficile Toxin B Shows Efficacy in Preventing Recurrent C. difficile Infection (CDI) in Patients at High Risk of Recurrence or of CDI-Related Adverse Outcomes.</a> Gastroenterology. 150 (4), S122 (2016).</li><li>Tsutsumi, L. S., Owusu, Y. B., Hurdle, J. G., Sun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+discovery+of+treatments+for+C.+difficile+infection:+A+clinical+and+medicinal+chemistry+review.">Progress in the discovery of treatments for C. difficile infection: A clinical and medicinal chemistry review.</a> Curr Top Med Chem. 14 (1), 152-175 (2014).</li><li>Hensbergen, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+secreted+Pro-Pro+endopeptidase+PPEP-1+(ZMP1/CD2830)+modulates+adhesion+through+cleavage+of+the+collagen+binding+protein+CD2831.">Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831.</a> FEBS Lett. 589 (24), 3952-3958 (2015).</li><li>Cafardi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+zinc+metalloprotease+through+a+global+analysis+of+Clostridium+difficile+extracellular+proteins.">Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins.</a> PLoS One. 8 (11), e81306 (2013).</li><li>Hensbergen, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+secreted+metalloprotease+(CD2830)+from+Clostridium+difficile+cleaves+specific+proline+sequences+in+LPXTG+cell+surface+proteins.">A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.</a> Mol Cell Proteomics. 13 (5), 1231-1244 (2014).</li><li>Schacherl, M., Pichlo, C., Neundorf, I., Baumann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+of+Proline-Proline+Peptide+Bond+Specificity+of+the+Metalloprotease+Zmp1+Implicated+in+Motility+of+Clostridium+difficile.">Structural Basis of Proline-Proline Peptide Bond Specificity of the Metalloprotease Zmp1 Implicated in Motility of Clostridium difficile.</a> Structure. 23 (9), 1632-1642 (2015).</li><li>Rawlings, N. D., Waller, M., Barrett, A. J., Bateman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEROPS:+the+database+of+proteolytic+enzymes,+their+substrates+and+inhibitors.">MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.</a> Nucleic Acids Res. 42 (Release 10.0), D503-D509 (2014).</li><li>Bergfors, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeds+to+crystals.">Seeds to crystals.</a> J Struct Biol. 142 (1), 66-76 (2003).</li><li>Dauter, Z., Dauter, M., Dodson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jolly+SAD.">Jolly SAD.</a> Acta Crystallogr D Biol Crystallogr. 58 (Pt 3), 494-506 (2002).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 2), 125-132 (2010).</li><li>Adams, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallogr D Biol Crystallogr. 66, 213-221 (2010).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 4), 486-501 (2010).</li><li>. <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 Graphics System. , (2002).</li><li>Pettersen, E. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=His-tag+impact+on+structure.">His-tag impact on structure.</a> Acta Crystallogr D Biol Crystallogr. 63 (Pt 3), 295-301 (2007).</li><li>Gasteiger, E., Walker, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Identification+and+Analysis+Tools+on+the+ExPASy+Server.">Protein Identification and Analysis Tools on the ExPASy Server.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Dummler, A., Lawrence, A. 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The authors have nothing to disclose.

X-ray crystallography is still the fastest and most accurate method to determine three-dimensional near-atomic resolution structures of proteins28. However, it requires the growth of well-ordered single crystals. These are often difficult to get and the crystalline state is artificial. However, a comparison of protein structures determined by X-ray crystallography with those determined by other methods, especially NMR, shows generally a very good agreement. In the case of PPEP-1, an NMR structure published rec...

Clostridium difficile is one of the major causes of nosocomial antibiotic-associated diarrhea infections1. This Gram-positive anaerobic bacterium is transmitted through its spore form via the fecal-oral route. In the past decade, new &#39;&#39;epidemic&#39;&#39; or &#39;&#39;hypervirulent&#39;&#39; strains (e.g. BI/NAP1/027) caused a drastic increase in new infections and fatality rates in North America and Europe2. C. difficile-associated disease (CDAD) is a life threatening colon inflammation with high fatality rates3. The symptoms range from diarrhea4 to pseudomembranous colitis5 and the often-fatal toxic megacolon6.
Treatment of CDAD is difficult as the virulent strains are multidrug-resistant and the recurrence rate is high7. At the moment therapy includes the antibiotics metronidazole, fidaxomicin or vancomycin, or in repetitively recurrent cases fecal microbiota transplantation. New therapeutic strategies are urgently needed8. Some progress is recorded as the therapeutic monoclonal antibody Bezlotoxumab, targeting C. difficile toxin B9, has recently successfully passed phase III clinical trials and was filed for approval with the FDA and EMA. Additionally, new antibiotics are being tested at the moment at different stages of clinical trials10.
To develop effective treatment new therapeutic targets must be identified. The recently discovered C. difficile protease proline-proline endopeptidase-1 (PPEP-1; CD2830/Zmp1; E.C. 3.4.24.89) is such a promising target, as the lack of PPEP-1 in a knock-out strain decreases virulence of C. difficile in vivo11. PPEP-1 is a secreted metalloprotease12,13 cleaving two C. difficile adhesins at their C-terminus13 thus releasing the adherent bacteria from the human gut epithelium. Therefore, it is involved in maintaining the balance between the sessile and motile phenotype of C. difficile. To develop selective inhibitors against PPEP-1 and to understand how it recognizes its substrates intimate knowledge of its three-dimensional structure is indispensable. We have solved the first crystal structure of PPEP-1 alone and in complex with a substrate peptide14. PPEP-1 is the first known protease that selectively cleaves peptide bonds between two proline residues15. It binds the substrate in a double-kinked manner and stabilizes it via an extended aliphatic-aromatic network of residues located in the S-loop that covers the protease active site14. This substrate-binding mode is unique to PPEP-1 and not found in human proteases to date. This makes it a promising drug target, and off-target effects of inhibitors very unlikely.
To develop and screen selective PPEP-1 inhibitors in the future a large amount of pure and monodisperse PPEP-1 protein is needed. Furthermore, to determine the mode of binding of first inhibitors, co-crystal structures with PPEP-1 will have to be determined. In our hands PPEP-1 constantly produces intergrown crystals. Thus we have developed an optimization procedure to produce single diffraction-quality crystals of PPEP-1. In this protocol we describe in detail the production, purification, crystallization and structure solution of PPEP-114. We use intracellular expression in Escherichia coli of a PPEP-1 variant lacking the secretion signal sequence, affinity chromatography and size exclusion chromatography with removal of the purification tag, followed by microseeding16 into an optimization screen and structure determination via zinc single-wavelength anomalous dispersion (zinc-SAD)17. This protocol can be adapted for production and structure determination of other proteins (e.g. metalloproteases) and in particular for proteins producing intergrown crystals. On request, plasmid DNA of the construct (pET28a-NHis-rPPEP-1) and diffraction data can be provided for educational purposes.

<table border="0" cellpadding="0" cellspacing="0" width="657">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Genes / Vectors / cell strains</td>
			<td style="width:83px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET28a vector</td>
			<td>Merck-Millipore</td>
			<td>69864</td>
			<td>Thrombin cleavable N-terminal His-tag</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E. coli strain BL21 (DE3) Star</td>
			<td>ThermoFisher Scientific</td>
			<td>C601003</td>
			<td>RNase H deficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Codon-optimized gene (for E. coli) of PPEP-1 (CD630_28300)</td>
			<td>Geneart (Thermo Fisher Scientific)</td>
			<td>custom</td>
			<td>amino acids 27-220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast extract</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antifoam B</td>
			<td>Sigma-Aldrich</td>
			<td>A5757</td>
			<td>aqueous-silicone emulsion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>AppliChem</td>
			<td>A1008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>AppliChem</td>
			<td>A1087</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>any</td>
			<td></td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNaseI</td>
			<td>AppliChem</td>
			<td>A3778</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>AppliChem</td>
			<td>A1073</td>
			<td>Buffer grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>215996</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol 100%</td>
			<td>any</td>
			<td></td>
			<td>purest grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid nitrogen</td>
			<td>any</td>
			<td></td>
			<td>for storage and cryocooling of crystals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (general)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking incubator</td>
			<td>any</td>
			<td></td>
			<td>providing temperatures of 20 &#176;C - 37 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glassware</td>
			<td>any</td>
			<td></td>
			<td>baffled Erlenmeyer flasks (50 ml - 2.8L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge for large culture volumes</td>
			<td>any</td>
			<td></td>
			<td>centrifuge for processing volumes up to 12 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator Vibra-Cell VCX500</td>
			<td>Sonics</td>
			<td>SO-VCX500</td>
			<td>or any other sonicator / cell disruptor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultracentrifuge</td>
			<td>any</td>
			<td></td>
			<td>centrifuge providing speeds up to 150.000 x g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NiNTA Superflow resin</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Empty Glass Econo-Column</td>
			<td>Bio-Rad</td>
			<td>7371007</td>
			<td>or any other empty glass or plastic column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Size exclusion chromatography column HiLoad Superdex 200 16/600</td>
			<td>GE Healthcare</td>
			<td style="width:136px;">28989335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chromatography system &#196;kta Purifier</td>
			<td>GE Healthcare</td>
			<td>28406264</td>
			<td>or any other chromatography system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing Spectra/Por 3</td>
			<td>Spectrum Labs</td>
			<td>132724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dialysis tubing closures</td>
			<td>Spectrum Labs</td>
			<td>132738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrafiltration units (concentrators) 10.000 NWCO</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis spectrophotometer</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment (crystallography)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Low volume pipette 0.1-10 &#181;l</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Positive displacement pipette Microman M10</td>
			<td>Gilson</td>
			<td>F148501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystallization robot</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well crystallization plates TTP IQ with three protein wells</td>
			<td>TTP</td>
			<td style="width:136px;">4150-05810</td>
			<td>or any other 96-well crystallization plate&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well CombiClover Junior Plate</td>
			<td>Jena Bioscience</td>
			<td>EB-CJR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Crystal Clear Sealing Tape</td>
			<td>Hampton Research</td>
			<td>HR3-511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Siliconized Glass Cover Slides</td>
			<td>Hampton Research</td>
			<td>HR3-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Commercial crystallization screens: Wizard, PACT++, JCSG++</td>
			<td>Jena Bioscience</td>
			<td>diverse</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JBS Beads-for-Seeds</td>
			<td>Jena Bioscience</td>
			<td>CO-501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalCap SPINE HT (nylon loops)</td>
			<td>Hampton Research</td>
			<td>diverse</td>
			<td>loop sizes 0.025 mm - 0.5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalCap Vial</td>
			<td>Hampton Research</td>
			<td>HR4-904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 800 ml</td>
			<td>Hampton Research</td>
			<td>HR4-673</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Cryogenic Foam Dewar 2L</td>
			<td>Hampton Research</td>
			<td>HR4-675</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vial Clamp, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-670</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CrystalWand Magnetic, Straight</td>
			<td>Hampton Research</td>
			<td>HR4-729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane 6 Vial Holder</td>
			<td>Hampton Research</td>
			<td>HR4-711</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoSleeve</td>
			<td>Hampton Research</td>
			<td>HR4-708</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoCane Color Coder - White</td>
			<td>Hampton Research</td>
			<td>HR4-713</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight microforcep</td>
			<td>any</td>
			<td></td>
			<td>for manipulation of sealing tape. etc.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acupuncture needle</td>
			<td>any</td>
			<td></td>
			<td>e.g. from a pharmacy</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Stereo microscope</td>
			<td>any</td>
			<td></td>
			<td style="width:191px;">for inspection of crystallization plates and crystal mounting, magnification up to 160X</td>
		</tr>
	</tbody>
</table>

production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

rPPEP-1 is overexpressed in several E. coli strains, with the highest yield in E. coli BL21 (DE3) Star (Figure 1C). After the first NiNTA affinity chromatography step the 6xHis-tag can be successfully cleaved off from most of the protein and in the second NiNTA step undigested protein can be completely separated from thrombin-digested protein (Figure 1D). On a S200 16/600 column untagged rPPEP-1 migrates as monomer with occasional fronti...

Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

production-crystallization-structure-determination-c-difficile-ppep-1

Research

JoVE Journal

Biochemistry

11.5K Views.  University of Cologne. Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein. 

Video: Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 &#197; resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin&#8211;mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin&#8211;mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin&#8211;mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR&#8211;G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.

This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since ...