We thank Prof. D. Madden, for many helpful discussions throughout this project. We thank Prof. D. Bolon for the gift of the plasmid containing the optimized GCN4 coiled coil. We thank Dr. B. Guo for NEMO plasmids. We thank Christina R. Arnoldy, Tamar Basiashvili and Amy E. Kennedy for demonstrating the procedure. We thank the BioMT Crystallography Core Facility and the departments of Chemistry and of Biochemistry &#38; Cell Biology at Dartmouth for the use of the crystallography equipment and the BioMT personnel for their support. This research used the AMX beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. We thank the staff at NSLS II for their support. This work was funded by NIH grants R03AR066130, R01GM133844, R35GM128663 and P20GM113132, and a Munck-Pfefferkorn Novel and Interactive grant.

<ol>
	<li>Gilmore, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+NF-kappaB:+players,+pathways,+perspectives.">Introduction to NF-kappaB: players, pathways, perspectives.</a> Oncogene. 25 (51), 6680-6684 (2006).</li><li>Bass&#232;res, D. S., Baldwin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+factor-kappaB+and+inhibitor+of+kappaB+kinase+pathways+in+oncogenic+initiation+and+progression.">Nuclear factor-kappaB and inhibitor of kappaB kinase pathways in oncogenic initiation and progression.</a> Oncogene. 25 (51), 6817-6830 (2006).</li><li>Hayden, M. S., West, A. P., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+and+the+immune+response.">NF-kappaB and the immune response.</a> Oncogene. 25 (51), 6758-6780 (2006).</li><li>Lee, T. I., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+and+its+misregulation+in+disease.">Transcriptional regulation and its misregulation in disease.</a> Cell. 152 (6), 1237-1251 (2013).</li><li>Courtois, G., Gilmore, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+NF-kappaB+signaling+pathway:+implications+for+human+disease.">Mutations in the NF-kappaB signaling pathway: implications for human disease.</a> Oncogene. 25 (51), 6831-6843 (2006).</li><li>Zhao, 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=Development+of+novel+NEMO-binding+domain+mimetics+for+inhibiting+IKK/NF-&#954;B+activation.">Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-&#954;B activation.</a> PLoS biology. 16 (6), 2004663 (2018).</li><li>Zhang, Q., Lenardo, M. J., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=30+Years+of+NF-&#954;B:+a+blossoming+of+relevance+to+human+pathobiology.">30 Years of NF-&#954;B: a blossoming of relevance to human pathobiology.</a> Cell. 168 (1-2), 37-57 (2017).</li><li>Jin, D. Y., Jeang, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+full-length+cDNA+and+chromosomal+localization+of+human+NF-kappaB+modulator+NEMO+to+Xq28.">Isolation of full-length cDNA and chromosomal localization of human NF-kappaB modulator NEMO to Xq28.</a> Journal of Biomedical Science. 6 (2), 115-120 (1999).</li><li>Rushe, 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=Structure+of+a+NEMO/IKK-associating+domain+reveals+architecture+of+the+interaction+site.">Structure of a NEMO/IKK-associating domain reveals architecture of the interaction site.</a> Structure. 16 (5), 798-808 (2008).</li><li>Guo, B., Audu, C. O., Cochran, J. C., Mierke, D. F., Pellegrini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+of+the+N-terminus+of+NEMO:+structure+stabilization+and+rescue+of+IKK&#946;+binding.">Protein engineering of the N-terminus of NEMO: structure stabilization and rescue of IKK&#946; binding.</a> Biochemistry. 53 (43), 6776-6785 (2014).</li><li>Havranek, J. J., Harbury, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+design+of+specificity+in+molecular+recognition.">Automated design of specificity in molecular recognition.</a> Nature Structural Biology. 10 (1), 45-52 (2003).</li><li>Barczewski, A. H., Ragusa, M. J., Mierke, D. F., Pellegrini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IKK-binding+domain+of+NEMO+is+an+irregular+coiled+coil+with+a+dynamic+binding+interface.">The IKK-binding domain of NEMO is an irregular coiled coil with a dynamic binding interface.</a> Scientific Reports. 9 (1), 2950 (2019).</li><li>Arimori, T., 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=Fv-clasp:+an+artificially+designed+small+antibody+fragment+with+improved+production+compatibility,+stability,+and+crystallizability.">Fv-clasp: an artificially designed small antibody fragment with improved production compatibility, stability, and crystallizability.</a> Structure. 25 (10), 1611-1622 (2017).</li><li>Hernandez Alvarez, B., Hartmann, M. D., Albrecht, R., Lupas, A. N., Zeth, K., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+expression+system+for+protein+crystallization+using+trimeric+coiled-coil+adaptors.">A new expression system for protein crystallization using trimeric coiled-coil adaptors.</a> Protein Engineering, Design and Selection. 21 (1), 11-18 (2008).</li><li>Oshaben, K. M., Salari, R., McCaslin, D. R., Chong, L. T., Horne, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+native+GCN4+leucine-zipper+domain+does+not+uniquely+specify+a+dimeric+oligomerization+state.">The native GCN4 leucine-zipper domain does not uniquely specify a dimeric oligomerization state.</a> Biochemistry. 51 (47), 9581-9591 (2012).</li><li>Truebestein, L., Leonard, 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=Coiled-coils:+The+long+and+short+of+it.">Coiled-coils: The long and short of it.</a> BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology. 38 (9), 903-916 (2016).</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> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Kapust, R. B., 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=Tobacco+etch+virus+protease:+mechanism+of+autolysis+and+rational+design+of+stable+mutants+with+wild-type+catalytic+proficiency.">Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.</a> Protein Engineering. 14 (12), 993-1000 (2001).</li><li>Miladi, B., 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+new+tagged-TEV+protease:+construction,+optimisation+of+production,+purification+and+test+activity.">A new tagged-TEV protease: construction, optimisation of production, purification and test activity.</a> Protein Expression and Purification. 75 (1), 75-82 (2011).</li><li>Miller, M. S., 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=Getting+the+Most+Out+of+Your+Crystals:+Data+Collection+at+the+New+High-Flux,+Microfocus+MX+Beamlines+at+NSLS-II.">Getting the Most Out of Your Crystals: Data Collection at the New High-Flux, Microfocus MX Beamlines at NSLS-II.</a> Molecules. 24 (3), (2019).</li><li>McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phaser+crystallographic+software.">Phaser crystallographic software.</a> Journal of Applied Crystallography. 40, 658-674 (2007).</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 Crystallographica Section D: Biological Crystallography. 66, 213-221 (2010).</li><li>Terwilliger, T. C., 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=Iterative+model+building,+structure+refinement+and+density+modification+with+the+PHENIX+AutoBuild+wizard.">Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.</a> Acta Crystallographica. Section D, Biological Crystallography. 64, 61-69 (2008).</li><li>Emsley, P., 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=Coot:+model-building+tools+for+molecular+graphics.">Coot: model-building tools for molecular graphics.</a> Acta Crystallographica. Section D, Biological Crystallography. 60, 2126-2132 (2004).</li><li>Terwilliger, T. C., 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.mr_rosetta:+molecular+replacement+and+model+rebuilding+with+Phenix+and+Rosetta.">phenix.mr_rosetta: molecular replacement and model rebuilding with Phenix and Rosetta.</a> Journal of Structural and Functional Genomics. 13 (2), 81-90 (2012).</li><li>Strong, M., Sawaya, M. R., Wang, S., Phillips, M., Cascio, D., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+structural+genomics+of+complexes:+crystal+structure+of+a+PE/PPE+protein+complex+from+Mycobacterium+tuberculosis.">Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (21), 8060-8065 (2006).</li><li>Tickle, I. 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> STARANISO. , (2018).</li><li>French, S., Wilson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+treatment+of+negative+intensity+observations.">On the treatment of negative intensity observations.</a> Acta Crystallographica Section A: Crystal Physics, Diffraction, Theoretical and General Crystallography. 34 (4), 517-525 (1978).</li><li>Goldschmidt, L., Cooper, D. R., Derewenda, Z. S., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+rational+protein+crystallization:+A+Web+server+for+the+design+of+crystallizable+protein+variants.">Toward rational protein crystallization: A Web server for the design of crystallizable protein variants.</a> Protein Science: A Publication of the Protein Society. 16 (8), 1569-1576 (2007).</li><li>Zhou, L., 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=Disulfide-mediated+stabilization+of+the+I&#954;B+kinase+binding+domain+of+NF-&#954;B+essential+modulator+(NEMO).">Disulfide-mediated stabilization of the I&#954;B kinase binding domain of NF-&#954;B essential modulator (NEMO).</a> Biochemistry. 53 (50), 7929-7944 (2014).</li><li>D'Arcy, A., Bergfors, T., Cowan-Jacob, S. W., Marsh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microseed+matrix+screening+for+optimization+in+protein+crystallization:+what+have+we+learned.">Microseed matrix screening for optimization in protein crystallization: what have we learned.</a> Acta Crystallographica. Section F, Structural Biology Communications. 70, 1117-1126 (2014).</li></ol>

The authors declare no competing interests.

Crystallization attempts of NEMO in the unbound form were unsuccessful, including attempts using the full-length protein and several truncation constructs encompassing the IKK-binding domain. Our biophysical characterization of the IKK-binding domain of NEMO (residues 44-111) by circular dichroism, NMR spectroscopy and fluorescence anisotropy indicated that the construct, albeit able to bind IKK&#946;, existed in a state of conformational exchange, not suitable for crystallization9,10. Our approach involved engineering a construct of the IKK-binding domain of NEMO that adopted a more stable, folded and native-like conformation, eliminating the flexibility that prevented crystallization. Ideal coiled-coil domain adaptors fused to the N- and C-termini of the NEMO(44-111) sequence offer the advantage of stabilizing the dimeric coiled-coil fold of the native NEMO and facilitating crystallization14. Behavior of a series of protein constructs was monitored at each step through SDS-PAGE, size exclusion chromatography, circular dichroism, fluorescence anisotropy and NMR spectroscopy, as described earlier10,12. Despite the progressive improvement in all desired parameters no construct yielded diffraction quality crystals until the mutations E56A, E57A were introduced. The mutations were the highest impact mutations suggested by the Surface Entropy Reduction Server SERp29 that did not involve residues implicated in hot-spots of binding for IKK&#946;, as in the NEMO / IKK&#946; complex structure. Although other constructs stabilizing the ordered structure of the IKK-binding domain of NEMO through cysteine disulfide linkage and binding IKK&#946; with high affinity have been described30, the protocol here described represents the first successful approach to structure determination of the IKK-binding domain of NEMO in the unbound form.
Protein production and purification followed standard procedures in our laboratory. Upon cell lysis a considerable portion of the protein is present in the insoluble fraction, even when growing the cells after induction at 18 &#176;C, therefore the protein was solubilized in 8 M urea after cell lysis and prior to purification, and refolded while bound to the IMAC column. Extensive washing of the column bound protein both under denaturing conditions and after refolding are critical for a pure product after the first purification step. The pure NEMO-EEAA has a higher tendency to precipitate than the previous constructs but is still sufficiently soluble under crystallization conditions.
A critical step in the structure determination was the use of seeding. In an approach similar to microseed matrix screening31, seeds from crystal obtained during sparse matrix screening were used in an additional screening of conditions, followed by a fine screening round, in order to determine final crystallization conditions. Most of the crystals grown in the final conditions display high mosaicity and are not suitable for data collection. Crystals were screened over several visits to the beamline and best data quality was achieved by collecting datasets at the edge of the crystal, where the newest growth had occurred. As the region which showed lattice uniformity in cross-polarized images was small, it was instrumental to have access to the AMX beamline at NSLS II, due to the small beam size.
We successfully determined the structure of NEMO-EEAA by molecular replacement using as a search model the structure of the dimeric coiled-coil of GCN415, which mapped to the ideal coiled-coil adaptors fused at the N- and C-termini of the NEMO sequence. The molecular replacement was successful as the GCN4 adaptors maintain their native structure when fused to NEMO. At the same time, we could verify that NEMO maintains its native like structure by comparing the portions not involved in IKK-binding with the corresponding structure region in the NEMO / IKK&#946; complex structure. As an alternative, it could be possible to use the structure of NEMO in the NEMO / IKK&#946; complex (PDB: 3BRV)9 as a molecular replacement search model, focusing on monomer B, which corresponds to the chain which undergoes the smallest conformational change upon ligand binding. This strategy showed some initial success using the MR-ROSETTA module of PHENIX.
Finally, it was essential for a successful structure determination to utilize all available data in the dataset, by resorting to an anisotropic cut-off of merged intensity data, as provided by the STARANISO27 server or the Diffraction Anisotropy Server at UCLA26.
The essential role played by the NEMO/IKK complex in the NF-kB pathway makes it a desirable target for modulation of the pathway for therapeutic intervention. Protein-protein interactions, especially when involving a large binding interface, are challenging to inhibit with small peptides or small molecules, and structure-based inhibitor design can offer a significant advantage. The constructs of the IKK-binding domain of NEMO that we developed overcome the limits of the flexible NEMO(44-111) to facilitate crystallization and structure determination. As the construct easily crystallizes in the unbound form, we envision that the same protocol can be successfully applied in the crystallization of the complex of NEMO with small molecule inhibitors, to determine a structure that would provide details on binding modes and allow further ligand improvements.
An analysis of crystal packing in the crystallization conditions achieved in this work indicates that ligand co-crystallization may be preferred to ligand soaking into apo-protein crystals. While crystal packing provides some space around chain B of the dimer (6 to 10 &#197; to the nearest chain) and on one face of the ligand binding site (approximately 13 &#197; in the hot-spot region between Phe82-Phe87), the symmetric binding pocket is completely occluded by nearby chains, preventing ligand binding.
Coiled coils are present in a significant proportion in the proteome and are essential in their function as molecular spacers, scaffolds and in communicating conformational changes16. Despite their abundance and relevant roles, there are relatively few structures of full-length coiled-coil proteins. The use of coiled-coil adaptors may aid in the stabilization of structural domains of coiled-coil proteins while preserving their native structure and aid in the structural determination and elucidation of their function.

The NF-&#954;B pathway is activated in response to a variety of stimuli, including cytokines, microbial products and stress, to regulate expression of target genes responsible for inflammatory and immune response, cell death or survival and proliferation1. Pathologies including inflammatory and autoimmune diseases and cancer2,3,4,5 have been correlated to hyperactivation of the pathway, which has made modulation of NF-&#954;B activity a prime target for the development of new therapies6,7.
The canonical NF-&#954;B pathway in particular is distinguished from the non-canonical pathway, responsible for lymphorganogenesis and B-cell activation, by the former&#39;s dependence on the scaffolding protein NEMO (NF-&#954;B essential modulator8) for the assembly of the IKK-complex with the kinases IKK&#945; and IKK&#946;. The IKK complex is responsible for the phosphorylation of I&#954;B&#945; (inhibitor of &#954;B) that targets it for degradation, freeing the NF-&#954;B dimers to translocate to the nucleus for gene transcription1 and is therefore an attractive target for the development of inhibitors to modulate NF-&#954;B activity.
Our research focuses on the characterization of the protein-protein interaction between NEMO and IKK&#946;, targeting NEMO for the development of small molecules inhibitors of IKK complex formation. The minimal binding domain of NEMO, required to bind IKK&#946;, encompasses residues 44-111, and its structure has been determined in complex with a peptide corresponding to IKK&#946; sequence 701-7459. NEMO and IKK&#946; form a four-helix bundle where the NEMO dimer accommodates the two helices of IKK&#946;(701-745) in an elongated open groove with an extended interaction interface. IKK&#946;(734-742), also known as the NEMO-binding domain (NBD), defines the most important hot-spot for binding, where the two essential tryptophans (739,741) bury deeply within the NEMO pocket. The details of the complex structure can aid in the structure-based design and optimization of small molecule inhibitors targeting NEMO. At the same time, it is difficult that binding of a small molecule or peptide would recreate in NEMO the full conformational change (i.e., extensive opening of the NEMO coiled-coil dimer) caused by binding of the long IKK&#946;(701-745), as observed in the crystal, and the structure of unbound NEMO or NEMO bound to a small molecule inhibitor may represent a better target for structure-based drug design and inhibitor optimization.
Full length NEMO and smaller truncation constructs encompassing the IKK-binding domain have proven intractable for structure determination in the unbound form via X-ray crystallography and nuclear magnetic resonance (NMR) methods10, which prompted us to design an improved version of the IKK-binding domain of NEMO. Indeed, NEMO (44-111) in the unbound form is only partially folded and undergoes conformational exchange and we therefore set to stabilize its dimeric structure, coiled-coil fold and stability, while preserving binding affinity for IKK&#946;. By appending three heptads of ideal dimeric coiled-coil sequences11 at the N-and C-termini of the protein, and a series of four point mutations, we generated NEMO-EEAA, a construct fully dimeric and folded in a coiled coil, which rescued IKK-binding affinity to the nanomolar range as observed for full length NEMO12. As an additional advantage, we hoped the coiled-coil adaptors (based on the GCN4 sequence) would facilitate crystallization and eventually aid in the X-ray structure determination via molecular replacement. Coiled-coil adaptors have been similarly utilized to both increase stability, improve solution behavior and facilitate crystallization for trimeric coiled coils and antibody fragments13,14. NEMO-EEAA is easily expressed and purified from Escherichia. coli cells with a cleavable Histidine tag, is soluble, folded in a stable dimeric coiled coil and is easily crystallized, with diffraction to 1.9 &#197;. The presence of the ordered coiled-coil regions of GCN4 could additionally aid in phasing the data from crystals of NEMO-EEAA by molecular replacement using the known structure of GCN415.
Given the results obtained with apo-NEMO-EEAA, we believe the protocols described here could also be applied to the crystallization of NEMO-EEAA in the presence of small peptides (like the NBD peptide) or small molecule inhibitors, with the goal of understanding the requirements for NEMO inhibition and structure-based optimization of initial lead inhibitors to high affinity. Given the plasticity and dynamic nature of many coiled-coil domains16, the use of the coiled-coil adaptors could find more general applicability in aiding structural determination.

<table><tbody><tr><td>20% w/v &amp;gamma;-PGA (Na+ form, LM)</td><td>Molecular Dimensions</td><td>MD2-100-150</td><td>For fine screen crystallization of NEMO-EEAA</td></tr><tr><td>3.5 kDa MWCO Dialysis Membrane</td><td>Spectra/Por</td><td>132724</td><td>For dialysis removal of imidazole</td></tr><tr><td>Amicon Stirred Cell</td><td>Millipose Sigma</td><td>UFSC 05001</td><td>For protein concentration</td></tr><tr><td>Ammonium Chloride</td><td>Millipore Sigma</td><td>G8270</td><td>For minimal media labeling</td></tr><tr><td>Benzonase Nuclease</td><td>Millipore Sigma</td><td>9025-65-4</td><td>For digestion of nucleic acid</td></tr><tr><td>BL21-CodonPlus (DE3)-RIL Competent Cells</td><td>Agilent Technologies</td><td>Model: 230245</td><td>TEV expression</td></tr><tr><td>CryoPro</td><td>Hampton Research</td><td>HR2-073</td><td>Cryo-protectants kit</td></tr><tr><td>D-Glucose (Dextrose)</td><td>Millipore Sigma</td><td>A9434</td><td>For minimal media labeling</td></tr><tr><td>Difco Terrific Broth</td><td>ThermoFisher</td><td>DF043817</td><td>For culture growth</td></tr><tr><td>Dithiothreitol &gt; 99%</td><td>Goldbio</td><td>DTT25</td><td>For reduction of disulfides</td></tr><tr><td>E. coli: Rosetta 2 (DE3)</td><td>Novagen</td><td>71400-3</td><td>Expression of unlabeled NEMO-EEAA</td></tr><tr><td>FORMULATOR</td><td>Formulatrix</td><td></td><td>Liquid handler/ screen builder</td></tr><tr><td>HCl &amp;ndash; 1.0 M Solution</td><td>Hampton Research</td><td>HR2-581</td><td>For fine screen crystallization of NEMO-EEAA</td></tr><tr><td>HiLoad 16/600 Superdex 75 pg</td><td>GE Healthcare</td><td>28989333</td><td>For size exclusion purification</td></tr><tr><td>HisTrap HP 5 mL column</td><td>GE Healthcare</td><td>17524802</td><td>For purification of His-tagged NEMO-EEAA</td></tr><tr><td>HT 96 MIDAS</td><td>Molecular Dimensions</td><td>MD1-59</td><td>For sparse matrix screening of NEMO-EEAA</td></tr><tr><td>HT 96 Morpheous</td><td>Molecular Dimensions</td><td>MD1-46</td><td>For sparse matrix screening of NEMO-EEAA</td></tr><tr><td>Imidazole</td><td>ThermoFisher</td><td>288-32-4</td><td>For elution from His-trap column</td></tr><tr><td>Isopropyl-beta-D-thiogalactoside</td><td>Goldbio</td><td>I2481C5</td><td>For induction of cultures</td></tr><tr><td>MRC2 crystallization plate</td><td>Hampton Research</td><td>HR3-083</td><td>Crystallization plate</td></tr><tr><td>NT8 - Drop Setter</td><td>Formulatrix</td><td></td><td>Crystallization</td></tr><tr><td>pET-16b</td><td>Millipore Sigma</td><td>69662</td><td>For cloning of NEMO-EEAA</td></tr><tr><td>pET-45b</td><td>Millipore Sigma</td><td>71327</td><td>For cloning of NEMO-EEAA</td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>ThermoFisher</td><td>36978</td><td>For inhibition of proteases</td></tr><tr><td>Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti</td><td>Beckmann Coulter</td><td>339160</td><td>Ultracentrifuge bottle</td></tr><tr><td>Polyethylene Glycol 20,000</td><td>Hampton Research</td><td>HR2-609</td><td>For fine screen crystallization of NEMO-EEAA</td></tr><tr><td>pRK793 (TEV)</td><td>Addgene</td><td>Plasmid 8827</td><td>For TEV production</td></tr><tr><td>QuikChange XL II</td><td>Agilent Technologies</td><td>200522</td><td>Site directed mutagenesis</td></tr><tr><td>Required Cap Assembly:</td><td>Beckmann Coulter</td><td>355623</td><td>Ultracenttrifuge bottle cap</td></tr><tr><td>ROCK IMAGER</td><td>Formulatrix</td><td></td><td>Crystallization Imager</td></tr><tr><td>Seed Bead Kit</td><td>Hampton Research</td><td>HR2-320</td><td>Seed generation</td></tr><tr><td>Sodium Chloride &amp;ge; 99%</td><td>Millipore Sigma</td><td>S9888</td><td>For buffering of purification solutions</td></tr><tr><td>TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)</td><td>Goldbio</td><td>TCEP1</td><td>Reducing agent</td></tr><tr><td>The Berkeley Screen</td><td>Rigaku</td><td>MD15-Berekely</td><td>For sparse matrix screening of NEMO-EEAA</td></tr><tr><td>The PGA Screen</td><td>Molecular Dimensions</td><td>MD1-50</td><td>For fine screen crystallization of NEMO-EEAA</td></tr><tr><td>Tris &amp;ndash; 1.0 M Solution</td><td>Hampton Research</td><td>HR2-589</td><td>For fine screen crystallization of NEMO-EEAA</td></tr><tr><td>Ultrapure Tris Buffer (powder format)</td><td>Thermofisher</td><td>15504020</td><td>For buffering of purification solutions</td></tr><tr><td>Urea</td><td>ThermoFisher</td><td>29700</td><td>For denaturation of NEMO-EEAA</td></tr></tbody></table>

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.

Cloning, expression and purification of the IKK-binding domain of NEMO. 
The protocol followed in this study to obtain the final sequence of NEMO-EEAA (Figure 1A), which produced diffraction quality crystals, involved the expression and characterization of all the intermediate constructs, including the addition of the coiled-coil adaptors at N- and or C-terminus, the mutations C76A, C95S and the mutations E56A, E57A. Figure 1B displays an SDS-PAGE gel of samples collected throughout the purification procedure as described in the Scheme in Figure 1C. The protein is overexpressed in E. coli and appears as a band approximately at the 14 kDa MW weight marker on the SDS-PAGE gel (lane 3, cells collected at harvest and lysed in Laemmli sample buffer supplemented by 8 M urea). The protein appears pure after the first IMAC column and displays a monomer and a dimer band at the level of the 14 and 28 kDa MW markers on the SDS-PAGE gel (lane 9). TEV cleavage is practically complete following the protocol and the protein elutes with the flow through during the second IMAC column almost entirely as a dimer, at the expected MW (band below 28 kDa). Size exclusion chromatography displays a single peak eluting between 60-65 mL and corresponding in our experience to the dimer (Figure 1D). The dimeric coiled coil always elutes earlier than expected on SEC due to the elongated shape of the coiled coil and the consequently large hydrodynamic radius10. NEMO-EEAA in fractions from SEC peak still appears as a monomer and a dimer on SDS-Page gel (lanes 14-15). Utilizing a stirred-cell concentrator is important to prevent sample possible aggregation and precipitation upon concentration.
Crystallization of NEMO-EEAA 
Initial crystals were obtained from a commercial screen using PGA (see Table of Materials), utilizing 1.65 &#181;g/mL of NEMO-EEAA in 2 mM Tris, 100 mM NaCl, 2 mM DTT, pH 8.0. Fine screening produced crystals in 0.1 M Tris pH 8.9, 5% PGA-LM, 3.6% PEG 20k (Figure 2A), which were utilized to produce a seed stock. Final crystals were obtained with seeding in 0.1 M Tris pH 8.0, 5.8% PGA-LM, 5.45% PEG 20k (Figure 2B).
Data collection and structure determination 
NEMO-EEAA crystals suffer from mosaicity and anisotropy. Crystals formed in the P 1 21 1 space group, with data resolution varying in the a*, b* and c* axis (1.88 &#197;, 2.10 &#197; and 2.55 &#197;). Examples of diffraction profiles are in Figure 3. Data was acquired at the AMX (17-ID-1) beamline of NSLS II, with a beam size of 7 x 5 &#956;m2. The small beam size was essential to focus on the desired portion of the crystal (Figure 2B) and ensure data quality.
The successful protocol for structure determination requires anisotropic truncation of the data using the STARANISO27 server (<a href="http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi" target="_blank">http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi</a>), followed by phasing by molecular replacement using MRage21 within PHENIX22 and the structure of dimeric GCN4 (PDB: 4DMD)15 as a search model. In the solution of this structure phasing was initially attempted by labeling the native methionine with SeMet. The anomalous signal was too weak, probably due to the solvent exposed nature of Met95 in the unbound form of NEMO (SeMet95 was successfully used for phasing of the NEMO / IKK&#946; structure9).
Initial data analysis was attempted with spherical truncation of the data to 2.3 &#197;. This data set could not be successfully phased by molecular replacement but a solution was obtained using MR-ROSETTA25 and the structure of NEMO(44-111) in complex with IKK&#946;(701-745) as a search model (PDB: 3BRV)9. This initial model could not be refined successfully. We utilized the Diffraction Anisotropy Server at UCLA26 to elliptically truncate the data and to remove anisotropy by anisotropic scaling. The newly processed dataset could be phased by molecular replacement. To further improve the data, we employed the STARANISO27 server for anisotropic truncation of the data and amplitudes were corrected with an anisotropic correction factor with Bayesian estimation28. This data, resulting in an increase in the number of unique reflections to 19,560, was used for structure refinement. Composite omit maps where calculated with PHENIX, excluding 10% of the atoms at a time, and compared with the final structure model, to confirm that the structure was not biased by the atomic model. The crystallographic model is complete except for the first and last residues in chain A of NEMO-EEAA, for which no electron density was observed.
NEMO-EEAA is a homo-dimeric, irregular, parallel coiled coil of ~175 &#197; in length. The regular coiled-coil region encompasses the ideal coiled-coil adaptor sequence at the N-terminus (residues 20-50) and the first two heptads of the NEMO proper sequence (residues 51-65). A regular coiled coil is also present at the C-terminus, starting at NEMO residue 97 and encompassing the C-terminal ideal coil-coil adaptor (Figure 4A). The central portion, encompassing NEMO residues 66-98 displays larger interhelical distances (interhelical spacing goes from an average value of 7.6 &#197; in the regular coiled-coil structure to a maximum of 11.5 &#197; in the irregular region) and discontinuous hydrophobic interface compared with an ideal coiled coil. This region of NEMO also represents the interface for binding IKK&#946; and undergoes a conformational change upon ligand binding. The IKK&#946;-bound structure displays a more open coiled-coil conformation to accommodate the ligand with larger interhelical spacing by 1.0 to 2.2 &#197; in this region (Figure 4B)12. In the apo structure residues Leu93, Met94, Lys96, Phe97 and Arg101 are shifted toward the center of the coiled coil to invade the ligand binding pocket (the C&#945;-C&#945; distance for Phe97 is tighter by 2.9 &#197;), with the overall effect of closing the large hydrophobic cleft which hosts the ligand in the IKK&#946;-bound structure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60339/60339fig1a.jpg" /> 
Figure 1: Purification of NEMO-EEAA. (A) Sequence alignment of NEMO from Homo Sapiens, Mus Musculus and Bos Bovis and the engineered NEMO-EEAA. The coiled-coil adaptors sequence is underlined, and the mutations are highlighted in orange. (B) SDS-PAGE gel of fractions collected during expression and purification (as labeled and referenced in flow chart 1C). 10% Acrylamide, MES buffer. (C) A flow chart for the purification of NEMO-EEAA. (D) Size-exclusion profile indicating the dimer of NEMO-EEAA (blue). In green the molecular weight markers (kDa) are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60339/60339fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60339/60339fig2a.jpg" /> 
Figure 2: Morphology and size of NEMO-EEAA crystals. (A) A crystal of NEMO-EEAA from the initial sparse matrix screen, unseeded. This type of crystal was used for seeding for subsequent crystallization attempts (0.1 M Tris pH 8.0, 5% PGA-LM, 3.6% PEG 20k; with a protein solution in 2 mM Tris, 100 mM NaCl 2 mM DTT, pH 8.0). (B) Crystal used for data collection (0.1 M Tris pH 8.0, 5.8% PGA-LM, 5.45% PEG 20k, with a protein solution in 2 mM Tris, 100 mM NaCl 2 mM DTT, pH 8.0). The approximate area used for data collection is circled in red. The scale bar for 100 &#181;m length is defined in the figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60339/60339fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60339/60339fig3a.jpg" /> 
Figure 3: Crystallographic data obtained from NEMO crystals. (A) X-ray diffraction profile of early NEMO crystal: elongated streaks indicate mosaicity in the crystal. (B) X-ray diffraction profile of an optimized NEMO-EEAA crystal. The resolution ring is drawn at a spatial resolution of 2.5 &#197;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60339/60339fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60339/60339fig4a.jpg" /> 
Figure 4: Structure of unbound NEMO. (A) The NEMO-EEAA dimer is shown as a ribbon, light blue = coiled-coil adaptors, blue = NEMO(51-112). (B) Superposition of apo NEMO-EEAA structure (blue, PDB: 6MI3) and NEMO(44-111) from the IKK&#946;-bound structure (grey, PDB: 3BRV, IKK&#946; is not displayed), shown as ribbons; the structures are aligned on chain A, region 44-111 only. This figure has been modified from previous publication12. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60339/60339fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO at JoVE.com

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

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 ...

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7.4K Views.  Dartmouth College. Dit protocol vergemakkelijkt de succesvolle structuurbepaling van het IKK bindingsdomein van NEMO, in zijn ongebonden vorm. En details van de productie en kristallisatie. Het protocol maakt gebruik van deze stabilisatie van de native bevestiging van het IKK bindingsdomeinfragment van NEMO, door middel van coiled-coil adapters, die kristallisatie en structuurbepaling vergemakkelijken.De structurele biologie van NEMO, als doelwit, biedt een belangrijk voordeel bij de ontwikkeling van NEM...

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

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

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 ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest control. To date, several diamide insecticides targeting pest RyRs have been commercialized, which generate annual revenue of 2 billion U.S. dollars. But comprehension of the mode of action of RyR-targeting insecticides is limited by the lack of structural information regarding insect RyR. This in turn restricts understanding of the development of insecticide resistance in pests. The diamondback moth (DBM) is a devastating pest destroying cruciferous crops worldwide, which has also been reported to show resistance to diamide insecticides. Therefore, it is of great practical importance to develop novel insecticides targeting the DBM RyR, especially targeting a region different from the traditional diamide binding site. Here, we present a protocol to structurally characterize the N-terminal domain of RyR from DBM. The x-ray crystal structure was solved by molecular replacement at a resolution of 2.84 &#197;, which shows a beta-trefoil folding motif and a flanking alpha helix. This protocol can be adapted for the expression, purification and structural characterization of other domains or proteins in general.

Crystal Structure of the N-terminal Domain of Ryanodine Receptor from Plutella xylostella

In this article, we describe the protocols of protein expression, purification, crystallization and structure determination of the N-terminal domain of&#160;ryanodine receptor from diamondback moth (Plutella xylostella).

Development of potent and efficient insecticides targeting insect ryanodine receptors (RyRs) has been of great interest in the area of agricultural pest ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

This article presents a method to generate protein crystals derivatized with I3C (5-amino-2,4,6-triiodoisophthalic acid) using microseeding to generate new crystallization conditions in sparse matrix screens. The trays can be set up using liquid dispensing robots or by hand.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Immunology and Infection

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

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

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