![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
Horizontal Tabs
Protein Expression and Purification
Preparation of the Human RIPK2 kinase domain
Boundaries: residues 3-317 (WT) or 8-317 (R171C mutant)
Vector: pFB-LIC-Bse
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: Sf9 insect cells
Bacmid DNA was prepared from DH10Bac cells and used to transfect Sf9 insect cells for the preparation of initial baculovirus. RIPK2 protein was expressed from infected Sf9 cells cultivated in InsectXpress medium (Lonza) for 48 hours at 27°C. Harvested cells were resuspended in lysis buffer (50 mM HEPES pH 7.4, 500 mM NaCl, 5 mM imidazole, 5% glycerol) supplemented 0.5 mM TCEP (tris(2-carboxyethyl)phosphine) and protease inhibitor cocktail set V (Calbiochem) at 1:1000 dilution. Cells were lysed using an Emulsiflex C5 homogeniser or by sonication, clarified by centrifugation, and the recombinant protein collected by nickel-affinity chromatography and eluted by imidazole. The tag was cleaved by TEV protease and RIPK2 protein either left untreated (heterogeneous 2-5 phosphorylations) or treated with lambda phosphatase overnight at 4°C. Further purification was performed on a Superdex 75 26/60 gel filtration column pre-equilibrated in 10 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, 1 mM TCEP. When dephosphorylated the final protein was prone to aggregation and was therefore supplemented with 5 mM L-arginine, 5 mM L-glutamate and 2 mM DTT.
Preparation of the Human XIAP BIR2 domain
Boundaries: residues 124-242 (Wild type or D214S mutant)
Vector: pGTVL2
Tag and additions: TEV-cleavable N-terminal hexahistidine and GST tags
Expression cell: E. coli (BL21DE3) cells
The WT or mutated (D214S) BIR2 domain of XIAP was expressed in E. coli (BL21DE3) cells as a fusion protein with N-terminal 6xHis and GST tags. Protein expression was induced with 0.5 mM IPTG for 16 h at 16 °C. Cells were lysed in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole, 0.5 mM TCEP) using an Emulsiflex C5 homogeniser or by sonication. After centrifugation at 4 °C the extract was loaded onto a 5 mL HisTrap FF column, washed and eluted with elution buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole. Further clean up was performed on a gel filtration column HiLoad 26/600 Superdex 75 pg (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 300 mM NaCl, 5% Glycerol, 0.5 mM TCEP. Collected fractions corresponding to monomeric form of the recombinant protein were frozen in liquid nitrogen, stored at -80 °C and used for probing of peptide array membranes. Alternatively, cells were lysed using BugBuster protein extraction reagent (#70921, Merck Millipore) and XIAP protein purified in batch using Glutathione Sepharose Fast Flow beads (GE Healthcare Life Sciences, Little Chalfont, UK). After 4 h, the beads were washed with PBS and directly used for GST pull-down experiments or stored at 4 °C.
Structure Determination
RIPK2 kinase domain with ponatinib (PDB: 4C8B)
Dephosphorylated RIPK2 R171C was concentrated to 3.7 mg/mL and ponatinib added to a slight molar excess. Crystals were grown using the vapour-diffusion technique in 150 nL sitting drops containing 75 nL protein and 75 nL of a reservoir solution containing 0.1 M ammonium citrate and 16% (w/v) PEG3350 at 20°C. Crystals were cryo-protected by addition of 25% ethylene glycol before being vitrified in liquid nitrogen. Diffraction data were collected at 100K on Diamond Light Source beamline I04. Data were indexed and integrated using XDS (16) and scaled using AIMLESS (17) in the CCP4 suite of programs (18). Phases were identified using molecular replacement in PHASER (19) and the PDB 3PPZ as a search model. Structures were built using PHENIX.AUTOBUILD (20) and then refined and modified using alternate rounds of REFMAC5 (21) and COOT (22). TLS groups were determined using the TLSMD server (23). The refined structure was validated with MolProbity (24).
RIPK2 kinase domain with CSR35 (PDB: 6ES0)
Phosphorylated WT RIPK2 was concentrated to 10 mg/mL in the presence of 2 mM CSR35 and 1% DMSO final concentration. After 10 min incubation, the mix was filtered to 0.22 µm. Crystals were grown using the vapour-diffusion technique in 150 nL sitting drops containing 50 nL protein and 100 nL of a reservoir solution containing 25% PEG Smear (low MW), 0.1 M MES pH 6.5, 0.05 M magnesium acetate, 0.05 M magnesium chloride at 20°C. Crystals were cryo-protected by addition of 25% ethylene glycol before being vitrified in liquid nitrogen. Diffraction data were collected at 100K on Diamond Light Source beamline I04. Data were indexed and integrated using XDS and scaled using AIMLESS. Phases were identified using molecular replacement in PHASER and the PDB ID: 4C8B as a search model. Structures were built using PHENIX.AUTOBUILD and then refined and modified using PHENIX.REFINE and COOT. The refined structure was validated with MolProbity and the atomic coordinate files deposited in the Protein Data Bank.
RIPK2 kinase domain with CSLP18 (PDB: 6FU5)
Phosphorylated WT RIPK2 was concentrated to 10 mg/mL in the presence of 2 mM CSLP18 and 1% DMSO final concentration. After 10 min incubation, the mix was filtered to 0.22 µm. Crystals were grown using the vapour-diffusion technique in 150 nL sitting drops containing 100 nL protein and 50 nL of a reservoir solution containing 0.2 M potassium formate and 20% (w/v) PEG3350 at 20°C. Crystals were cryo-protected by addition of 25% ethylene glycol before being vitrified in liquid nitrogen. Diffraction data were collected at 100K on Diamond Light Source beamline I04 using 0.9795 Å light. Data were indexed and integrated using XDS and scaled using AIMLESS. Phases were identified using molecular replacement in PHASER and the PDB ID: 4C8B as a search model. Structures were built using PHENIX.AUTOBUILD and then refined and modified using PHENIX.REFINE and COOT. The refined structure was validated with MolProbity and the atomic coordinate files deposited in the Protein Data Bank.
Assays
Analytical gel filtration
A Superdex 200 10/300 GL gel filtration column (GE healthcare) was attached to an Akta Purifier FPLC system (GE healthcare) and equilibrated in gel filtration buffer (50mM HEPES pH7.5, 150mM NaCl, 2mM TCEP). 500 μL of RIPK2 at 50 μM was injected onto the column, which was run at 0.5 mL/min and monitored using absorbance at 280 nm. The retention volume of RIPK2 was compared with the retention volumes of a set of gel filtration standards (Bio-Rad laboratories), which were run under the same conditions.
Thermal shift assay
A fluorescence-based thermal shift assay (differential scanning fluorimetry (DSF)) was performed as an initial screen to identify potential RIPK2 inhibitors. Ligands in this assay increase a protein’s melting temperature (Tm shift) by an amount proportional to their binding affinity. A solution of 2 µM RIPK2 protein in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5 mM TCEP, 5% glycerol) was mixed 1:1000 with SYPRO Orange (Sigma). Compounds to be tested were added to a final concentration of 10 µM. 20 µL of each sample were placed in a 96-well plate and heated from 25 to 95°C. Fluorescence was monitored using a Mx3005P real-time PCR instrument (Stratagene) with excitation and emission filters set to 465 and 590 nm, respectively. Data were analysed with the MxPro software and curves fit in Microsoft Excel using the Boltzmann equation to determine the midpoint of thermal denaturation (Tm). Thermal shift values (ΔTm) induced by inhibitor binding were calculated relative to control wells containing protein and 2.5% DMSO.
ADP-Glo in vitro kinase assays
For ADP-Glo (Promega) assays, 10 ng of RIPK2 was diluted in reaction buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mM DTT, 0.01% BSA) supplemented with 50 µM ATP and 10-point dose range of inhibitors. Reactions were performed at room temperature for 1 hour. Reactions were performed in 5 µL total volume (5% final concentration of DMSO) and stopped by addition of 5 µL of ADP-Glo reagent for 40 min at room temperature. Luminescent signal was generated by addition of 10 µL of kinase detection reagent for 30 minutes at room temperature and determined using Victor3V platereader (Perkin Elmer). Specific signal was calculated by subtracting values in the wells without protein and inhibitor from the values in the test wells. Inhibition, % = ((specific signal (DMSO control) - specific signal (inhibitor))/ (specific signal (DMSO control))) x 100%. Non-linear regression to determine IC50 values was performed using Prism software (GraphPad).
NOD2-HEK-Blue activation assay
For nanoBRET experiments full length RIPK2 was cloned into pFC32K-Nluc (Promega, Madison, WI). HEK-Blue hNOD2 cells (Invivogen) were maintained in DMEM medium (Fisher) supplemented with 10% FBS (Sigma) and 1% antibiotic-antimycotic mix (PSA) (Invitrogen) as well as Normocin (100 µg/mL), Blasticidin (30 μg/mL) and Zeocin (100 μg/mL). Cells (7.5x103 cells/mL) were seeded into clear 96 well plates and allowed to attach for 48 h. On the day of the experiment, media was changed to 100 μl of QUIATI-Blue detection media (InvivoGen). Inhibitors were diluted and added in 0.5 μL DMSO 15 min prior to the addition of 1 µg/mL L18-MDP (InvivoGen). After 8-10 hours, absorbance at 620 nM was determined using Victor3V plate reader (Perkin Elmer). Values of empty media were subtracted from all experimental samples. Resulting specific signal values were used to calculate inhibition, % = (1-(control (DMSO, L18-MDP) – sample (compound, L18-MDP))/(control (DMSO, L18-MDP)-control (DMSO)))*100. EC50 values were determined using non-linear regression in the Prism software package (GraphPad).
RIPK2 nanoBRET assay
HEK293 cells were cultured in DMEM GlutaMax (Gibco) supplemented with 10 % (v/v) FBS (Sigma-Aldrich) and 1 % (v/v) Penicillin-Streptomycin (Gibco). Cells were transiently transfected with a NanoLuc-RIPK2 construct together with Transfection Carrier DNA (Promega). After 20 h, transfected cells were plated at 2 × 105 cells/mL into a white 384-well assay plate (Corning, Corning, NY), treated with 0.25 µM fluorescent tracer SGC-590001, and incubated with inhibitor or DMSO control for 3 h. After addition of Nano-Glo Substrate (Promega) and Extracellular NanoLuc Inhibitor (Promega), BRET ratios (450 nm and 610 nm) were determined using a PHERAstar FSX plate reader (BMG Labtech). The preparation of SGC-590001 and further details of the assay were described previously (12).
NanoBRET inhibition assay
Inhibitor IC50 measurements were alternatively performed in HEKBlue cells. Cell density was adjusted to 2x105 cells/mL. 100x NanoBRET In-cell Kinase Tracer (Promega) was diluted to 20x in phenol red-free Opti-MEM supplemented with 12.5 mM HEPES (Thermo Fisher Scientific) and 31.25% PEG-400 (Sigma-Aldrich). 10x inhibitor stocks in Opti-MEM were prepared by diluting DMSO stocks. For the assay, 11.9 μL cells/well were seeded into a while low volume 384 well plate (Corning) and mixed with 0.7 μL 20x tracer and 1.4 μL 10x inhibitors, followed by incubation for 2 h at 37°C. 3x substrate mix was prepared by adding NanoBRET Nano-Glo substate (Promega) (1:166 dilution) and extracellular NanoLuc Inhibitor (Promega, 1:500 dilution) into Opti-MEM media. 7 μL of 3x substrate mix was added into each well with the cells. Plate was mixed on a rotary shaker for 15 sec at 500 rpm. Emission was determined using Victor3V plate reader at 460 nm for donor (NanoLuc) and 610 nm for acceptor (tracer). NanoBRET ratios were calculated as [(Acceptorsample / Donorsample) – (Acceptorno tracer control/Donorno tracer control)] x 1000 and used for non-linear regression to calculate IC50 values.
Immunoblot analysis
HEK-Blue cells were seeded into 10 cm2 dishes to achieve 80-90% confluency after 48 hr. Cells were pre-treated with inhibitors for 30 min and stimulated with L18-MDP for 30 min. Cells were lysed in RIPA buffer supplemented with PMSF), briefly sonicated and spun in a 4°C centrifuge at 14,000 rpm for 15 min to collect lysates. Protein concentrations were measured using 660 nM protein assay reagent (Pierce). Equal amounts of proteins were separated using 8-10% SDS-PAGE, followed by overnight incubations with antibodies according to the manufacturer’s recommendations. Commercially available antibodies included mouse anti-RIPK2 (clone A-10 from Santa Cruz), mouse anti-Ubiquitin (clone Ubi-1, Imgenex), rabbit anti-phospho-Ser176-RIPK2 (Cell Signaling), mouse anti-α-tubulin (clone DM1A, Cell Signaling), mouse anti-IĸBα (clone L35A5, Cell Signaling), and anti-actin (clone MAB1501, Millipore). The NOD1 ligand Tri-DAP, NOD2 ligands L18-MDP and MDP and TLR2 ligand Pam3CSK4 were purchased from Invivogen, LPS (E.coli 0111:B4) from Sigma and recombinant TNF from Enzo.
Analysis of RIPK2 ubiquitination in THP-1 cells
Human monocytic THP-1 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS and Penicillin / Streptomycin at density 0.6 −1.0 million cells per mL.. Cells were pretreated with kinase inhibitor or DMSO for 30 minutes and stimulated with 200 ng/mL L18-MDP for 45 mins to 4 hr. The ubiquitin conjugates were purified using GST-Tandem Ubiquitin Binding Entities (TUBE; (25)). Briefly, treated THP-1 cells (6−10×106) were washed with PBS and lysed in 400 µL of ice-cold lysis buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 1% NP-40, 2 mM EDTA) containing 50 μg/ml of GST-TUBE1 (Lifesensors, Malvern, PA) on ice for 30 minutes. Cleared lysates were incubated with Glutathione Sepharose 4B resin (Amersham) with agitation at 4°C overnight. The beads were washed four times with PBS-Tween (0.1%) and the bound proteins were released by heating the beads in reducing SDS sample buffer. The samples were subjected to immunoblotting using mouse anti-Ubiquitin (clone Ubi-1, Imgenex) and anti-RIPK2 antibodies.
Generation of RIPK2 KO U2OS/NOD2 cells and reconstitution with RIPK2 variants
NOD2-expressing U2OS-Flp-In™ T-REx™ (U2OS/NOD2) cells have been described previously (Fiil et al., 2013) and were cultured in DMEM GlutaMax (Gibco) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) Penicillin-Streptomycin (Gibco) and transfected using Fugene 6 (Promega). U2OS/NOD2 cells express doxycycline (DOX)-inducible HA-NOD2, but respond to L18-MDP without addition of DOX due to leakiness of the promoter (25). RIPK2 KO was generated using the CRISPR/Cas9 KO plasmids (containing gRNA, Cas9, and EGFP reporter) from Santa Cruz Biotechnology. These RIPK2 KO cells were then reconstituted retrovirally with WT RIPK2 or the kinase-dead RIPK2 mutants K47R or D146N (pBABE-Puro plasmids). All U2OS/NOD2 cells were cultured and stimulated in the absence of doxycycline unless otherwise indicated. The pcDNA3-HA-NOD2 expression vector was a gift to our collaborators from Dr G Nuñez (University of Michigan).
Dual luciferase NF-ĸB reporter assay
HEK293FT cells were co-transfected the NF-κB luciferase reporter construct pBIIX-luc and a thymidine kinase-renilla luciferase construct for normalization of transfection efficiency. Additionally, cells were co-transfected with WT or mutant HA-RIPK2 as required (pcDNA3-RIPK2-3xHA was a gift from T. Tenev/P. Meier). After 24-48 h, cells were lysed in 75 µL passive lysis buffer (Promega) and luciferase activity was measured on a FLUOstar Omega Microplate Reader (BMG LABTECH) using the Dual-Luciferase® Assay System (Promega). Where desired, cells were treated with DMSO or ponatinib (200 nM) 24 h before lysis.
SPOT synthesis peptide array
Cellulose-bound peptide arrays were prepared employing standard Fmoc solid phase peptide synthesis using a MultiPep-RSi-Spotter (INTAVIS, Köln, Germany) according to the manufacturer’s method. Arrays were probed with WT or D214S mutant 6xHIS-GST-tagged BIR2 domain of XIAP produced in E. coli. Bound protein was detected using anti-His antibody HPR conjugated (diluted 1:15000) and the Pierce® ECL Western blotting Substrate (#32106, Thermo Fisher Scientific). Chemiluminescence was detected using Super RX films (FUJIFILM) and quantification performed using ImageJ software.
GST-BIR2-XIAP pull down of cellular RIPK2 variants
For GST pull-down experiments, U2OS/NOD2 were lysed in TBS lysis buffer containing 0.5% NP-40, cOmplete and PhosSTOP inhibitors (Roche). Cleared lysates were pretreated with kinase inhibitors or DMSO and incubated with GST-XIAP-BIR2 bound to Glutathione Sepharose at 4 °C overnight. Bound material was washed 3x with lysis buffer or PBS, eluted with 15 mM Glutathione in PBS and analyzed by immunoblotting.
TNF ELISA Assay
RAW264.7 cells were maintained in DMEM media supplemented with 10 % heat-inactivated FBS and 1 % antibiotic-antimycotic mix (ThermoFisher Scientific). RAW264.7 cells were seeded into 12 well plates at a density of 1.5x105 cells/well in 1 mL of media. Inhibitors were diluted and added in 1 μL DMSO 15 min prior to the addition of 10 μg/mL MDP or 10 ng/mL E. coli LPS for 24 h. For TNF measurements, 100 μL of undiluted media (MDP) or 5-fold diluted media was analyzed using Duo-Set anti-mouse TNF ELISA kit (R&D Systems). Values of media-only wells were subtracted and %inhibition for each compound concentration relative to the DMSO/MDP (or LPS)-treated controls were calculated. IC50 values were calculated using Prism software (GraphPad).
- Girardin, S. E., Travassos, L. H., Herve, M., Blanot, D., Boneca, I. G., Philpott, D. J., Sansonetti, P. J., and Mengin-Lecreulx, D. (2003) Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2. The Journal of biological chemistry 278, 41702-41708
- Damgaard, R. B., Nachbur, U., Yabal, M., Wong, W. W., Fiil, B. K., Kastirr, M., Rieser, E., Rickard, J. A., Bankovacki, A., Peschel, C., Ruland, J., Bekker-Jensen, S., Mailand, N., Kaufmann, T., Strasser, A., Walczak, H., Silke, J., Jost, P. J., and Gyrd-Hansen, M. (2012) The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Molecular cell 46, 746-758
- Hugot, J. P., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J. P., Belaiche, J., Almer, S., Tysk, C., O'Morain, C. A., Gassull, M., Binder, V., Finkel, Y., Cortot, A., Modigliani, R., Laurent-Puig, P., Gower-Rousseau, C., Macry, J., Colombel, J. F., Sahbatou, M., and Thomas, G. (2001) Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 411, 599-603
- Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F., Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R. H., Achkar, J. P., Brant, S. R., Bayless, T. M., Kirschner, B. S., Hanauer, S. B., Nunez, G., and Cho, J. H. (2001) A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 411, 603-606
- Negroni, A., Stronati, L., Pierdomenico, M., Tirindelli, D., Di Nardo, G., Mancini, V., Maiella, G., and Cucchiara, S. (2009) Activation of NOD2-mediated intestinal pathway in a pediatric population with Crohn's disease. Inflamm Bowel Dis 15, 1145-1154
- Caso, F., Galozzi, P., Costa, L., Sfriso, P., Cantarini, L., and Punzi, L. (2015) Autoinflammatory granulomatous diseases: from Blau syndrome and early-onset sarcoidosis to NOD2-mediated disease and Crohn's disease. RMD Open 1, e000097
- Jurynec, M. J., Sawitzke, A. D., Beals, T. C., Redd, M. J., Stevens, J., Otterud, B., Leppert, M. H., and Grunwald, D. J. (2018) A hyperactivating proinflammatory RIPK2 allele associated with early-onset osteoarthritis. Hum Mol Genet
- Inaki, K., Menghi, F., Woo, X. Y., Wagner, J. P., Jacques, P. E., Lee, Y. F., Shreckengast, P. T., Soon, W. W., Malhotra, A., Teo, A. S., Hillmer, A. M., Khng, A. J., Ruan, X., Ong, S. H., Bertrand, D., Nagarajan, N., Karuturi, R. K., Miranda, A. H., and Liu, E. T. (2014) Systems consequences of amplicon formation in human breast cancer. Genome Res 24, 1559-1571
- Canning, P., Ruan, Q., Schwerd, T., Hrdinka, M., Maki, J. L., Saleh, D., Suebsuwong, C., Ray, S., Brennan, P. E., Cuny, G. D., Uhlig, H. H., Gyrd-Hansen, M., Degterev, A., and Bullock, A. N. (2015) Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors. Chem Biol 22, 1174-1184
- Nachbur, U., Stafford, C. A., Bankovacki, A., Zhan, Y., Lindqvist, L. M., Fiil, B. K., Khakham, Y., Ko, H. J., Sandow, J. J., Falk, H., Holien, J. K., Chau, D., Hildebrand, J., Vince, J. E., Sharp, P. P., Webb, A. I., Jackman, K. A., Muhlen, S., Kennedy, C. L., Lowes, K. N., Murphy, J. M., Gyrd-Hansen, M., Parker, M. W., Hartland, E. L., Lew, A. M., Huang, D. C., Lessene, G., and Silke, J. (2015) A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production. Nature communications 6, 6442
- Tigno-Aranjuez, J. T., Benderitter, P., Rombouts, F., Deroose, F., Bai, X., Mattioli, B., Cominelli, F., Pizarro, T. T., Hoflack, J., and Abbott, D. W. (2014) In vivo inhibition of RIPK2 kinase alleviates inflammatory disease. The Journal of biological chemistry 289, 29651-29664
- Vasta, J. D., Corona, C. R., Wilkinson, J., Zimprich, C. A., Hartnett, J. R., Ingold, M. R., Zimmerman, K., Machleidt, T., Kirkland, T. A., Huwiler, K. G., Ohana, R. F., Slater, M., Otto, P., Cong, M., Wells, C. I., Berger, B. T., Hanke, T., Glas, C., Ding, K., Drewry, D. H., Huber, K. V. M., Willson, T. M., Knapp, S., Muller, S., Meisenheimer, P. L., Fan, F., Wood, K. V., and Robers, M. B. (2018) Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell Chem Biol 25, 206-214 e211
- Mohedas, A. H., Wang, Y., Sanvitale, C. E., Canning, P., Choi, S., Xing, X., Bullock, A. N., Cuny, G. D., and Yu, P. B. (2014) Structure-activity relationship of 3,5-diaryl-2-aminopyridine ALK2 inhibitors reveals unaltered binding affinity for fibrodysplasia ossificans progressiva causing mutants. J Med Chem 57, 7900-7915
- Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H. W., Listgarten, J., and Root, D. E. (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34, 184-191
- Haile, P. A., Casillas, L. N., Bury, M. J., Mehlmann, J. F., Singhaus, R. Jr., Charnley, A. K., Hughes, T. V., DeMartino, M. P., Wang, G. Z., Romano, J. J., Dong, X., Plotnikov, N. V., Lakdawala, A. S., Convery, M. A., Votta, B. J., Lipshutz, D. B., Desai, B. M., Swift, B., Capriotti, C. A., Berger, S. B., Mahajan ,M. K., Reilly, M. A., Rivera, E. J., Sun, H. H., Nagilla, R., LePage, C., Ouellette, M. T., Totoritis, R. D., Donovan, B. T., Brown, B. S., Chaudhary, K. W., Gough, P. J., Bertin, J., Marquis, R. W. . (2018) Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel. ACS Med Chem Lett 9, 1039-1044
- Kabsch, W. (2010) Xds. Acta Crystallogr D Biol Crystallogr 66, 125-132
- Evans, P. R., and Murshudov, G. N. (2013) How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr 69, 1204-1214
- Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235-242
- McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J Appl Crystallogr 40, 658-674
- Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213-221
- Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr 67, 355-367
- Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501
- Painter, J., and Merritt, E. A. (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr D Biol Crystallogr 62, 439-450
- Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66, 12-21
- Fiil, B. K., Damgaard, R. B., Wagner, S. A., Keusekotten, K., Fritsch, M., Bekker-Jensen, S., Mailand, N., Choudhary, C., Komander, D., and Gyrd-Hansen, M. (2013) OTULIN restricts Met1-linked ubiquitination to control innate immune signaling. Molecular cell 50, 818-830
We respectfully request that this document is cited using the DOI value as given above if the content is used in your work.