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News Article | March 4, 2016
Site: www.nature.com

Mice were housed in the Unit for Laboratory Animal Medicine at the Whitehead Institute for Biomedical Research and Koch Institute for Integrative Cancer Research. The following strains were obtained from the Jackson Laboratory: Lgr5-EGFP-IRES-CreERT2 (strain name: B6.129P2-Lgr5tm1(cre/ERT2)Cle/J, stock number 008875), Rosa26-lacZ (strain name: B6.129S4-Gt(ROSA)26Sortm1Sor/J, stock number 003474), db/db (strain name: B6.BKS(D)-Leprjb/J, stock number 000697), PpardL/L (strain name: B6.129S4-Ppardtm1Rev/J, stock number 005897). Apcloxp exon 14 (ApcL/L) has been previously described41. Villin-CreERT2 was a gift from S. Robine. Long-term HFD was achieved by feeding male and female mice a dietary chow consisting of 60% kcal fat (Research Diets D12492) beginning at the age of 8–12 weeks and extending for a period of 9–14 months. Control mice were sex- and age-matched and fed standard chow ad libitum. GW501516 (Enzo) was reconstituted in DMSO at 4.5 mg ml−1 and diluted 1:10 in a solution of 5% PEG400 (Hampton Research), 5% Tween80 (Sigma), 90% H O for a daily intraperitoneal injection of 4 mg kg−1. Apc exon 14 was excised by tamoxifen suspended in sunflower seed oil (Spectrum S1929) at a concentration of 10 mg ml−1 and 250 μl per 25 g of body weight, and administered by intraperitoneal injection twice over 4 days before collecting tissue. PpardL/L mice were administered 4–5 intraperitoneal injections of tamoxifen on alternate days. Mice were analysed within 2 weeks of the last tamoxifen injection. BrdU was prepared at 10 mg ml−1 in PBS, passed through a 0.22-μm filter and injected at 100 mg kg−1. As previously described1, tissues were fixed in 10% formalin, paraffin embedded and sectioned. Antigen retrieval was performed with Borg Decloaker RTU solution (Biocare Medical) in a pressurized Decloaking Chamber (Biocare Medical) for 3 min. Antibodies used: rat anti-BrdU (1:2,000 (immunohistochemistry (IHC)), 1:1,000 (immunofluorescence (IF)) Abcam 6326), rabbit chromogranin A (1:4,000 (IHC), 1:250 (IF), Abcam 15160), rabbit monoclonal non-phospho β-catenin (1:800 (IHC), 1:400 (IF), CST 8814S), mouse monoclonal β-catenin (1:200, BD Biosciences 610154), rabbit polyclonal lysozyme (1:250, Thermo RB-372-A1), rabbit polyclonal MUC2 (1:100, Santa Cruz Biotechnology 15334), rabbit monoclonal OLFM4 (1:10,000, gift from CST, clone PP7), Biotin-conjugated secondary donkey anti-rabbit or anti-rat antibodies were used from Jackson ImmunoResearch. The Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs PK-6101) followed by Dako Liquid DAB+ Substrate (Dako) was used for visualization. For immunofluorescence, Alexa Fluor 568 secondary antibody (Invitrogen) was used with Prolong Gold (Life Technologies) mounting media. All antibody incubations involving tissue or sorted cells were performed with Common Antibody Diluent (Biogenex). Organoids were fixed with 4% paraformaldehyde, permabilized with 0.5% Triton X-100 in PBS, rinsed with 100 mM glycine in PBS, blocked with 10% donkey serum in PBS, incubated overnight with primary antibody at 4 °C, rinsed and incubated with Alexa Fluor 568 secondary antibody (Invitrogen), and mounted with Prolong Gold (Life Technologies) mounting media. The in situ hybridization probes used in this study correspond to expressed sequence tags or fully sequenced cDNAs obtained from Open Biosystems. The accession numbers (IMAGE mouse cDNA clone in parenthesis) for these probes are as follows: mouse Olfm4 BC141127 (9055739), mouse Crp4 BC134360 (40134597). Both sense and antisense probes were generated to ensure specificity by in vitro transcription using DIG RNA labelling mix (Roche) according to the manufacturer’s instructions and to previously published detailed methods23, 42. Single-molecule in situ hybridization was performed using Advanced Cell Diagnostics RNAscope 2.0 HD Detection Kit. Adult mice were exposed to 15 Gy of ionizing irradiation from a 137-caesium source (GammaCell) and euthanized after 72 h. The number of surviving crypts per length of the intestine was enumerated from haematoxylin-and-eosin-stained sections15. Antibodies: rabbit polyclonal anti-PPAR-δ (1:100, Thermo PA1-823A), rabbit polyclonal anti-CPT1a (1:250, ProteinTech 15184-1-AP), rabbit polyclonal anti-HMGCS2 (1:500, Sigma AV41562), rabbit monoclonal anti-FABP1 (1:1,000, Abcam ab129203), NF-κB Sampler Pathway Kit (CST, 9936S), mouse monoclonal anti-STAT-3 (CST, 9139P), rabbit monoclonal anti-P-STAT3 (Y705) XP (CST, 9145P), mouse monoclonal anti-CREB (CST, 86B10), mouse monoclonal anti-β-catenin (1:200, BD Biosciences 610154), rabbit polyclonal anti-γ-tubulin (1:1,000, Sigma T5192). For immunoprecipitation assays, crypts were collected and nuclear extraction was carried out using Abcam nuclear extraction kit (ab113474) following manufacturer’s instructions. Nuclear extracts were incubated with 5 μg anti-PPAR-δ antibody (Thermo), or anti-rabbit IgG control antibody (Santa Cruz) overnight at 4 °C followed by 2 h of incubation with Dynabeads Protein G for immunoprecipitation. Protein complexes bound to antibody and beads were washed five times and eluted with Laemmli sample buffer. Samples were resolved by SDS–PAGE. Protein interaction was analysed by immunoblotting. Lgr5-GFPhi ISCs or Lgr5-GFPlow progenitors were sorted directly into Laemmli sample buffer and boiled for 5 min. Samples were resolved by SDS–PAGE and analysed by immunoblotting with horseradish peroxidase (HRP)-conjugated IgG secondary antibodies (1:10,000, Santa Cruz Biotechnology sc-2054) and Western Lightning Plus-ECL detection kit (Perkin Elmer NEL104001EA) As previously reported and briefly summarized here, small intestines and colons were removed, washed with cold PBS without magnesium chloride and calcium (PBS−/−) opened longitudinally, and then cut into 3–5-mm fragments. Pieces were washed several times with cold PBS−/− until clean, washed 2–3 with PBS−/− EDTA (10 mM), incubated on ice for 90–120 min, and gently shook at 30-min intervals. Crypts were then mechanically separated from the connective tissue by more rigorous shaking, and then filtered through a 70-μm mesh into a 50-ml conical tube to remove villus material (for small intestine) and tissue fragments. Crypts were removed from this step for crypt culture experiments and embedded in Matrigel with crypt culture media. For ISC isolation, the crypt suspensions were dissociated to individual cells with TrypLE Express (Invitrogen). Cell labelling consisted of an antibody cocktail comprising CD45-PE (eBioscience, 30-F11), CD31-PE (Biolegend, Mec13.3), Ter119-PE (Biolegend, Ter119), CD24-Pacific Blue (Biolegend, M1/69), CD117-APC/Cy7 (Biolegend, 2BS), and EPCAM-APC (eBioscience, G8.8). ISCs were isolated as Lgr5-EGFPhiEpcam+CD24low/−CD31−Ter119−CD45−7-AAD−. EGFPlow progenitors were isolated as EGFPlowEpcam+CD24low/−CD31−Ter119−CD45−7-AAD−, and Paneth cells from small intestine were isolated as CD24hiSidescatterhiLgr5-EGFP−Epcam+CD31−Ter119−CD45−7-AAD− with a BD FACS Aria II SORP cell sorter into supplemented crypt culture medium for culture. Dead cells were excluded from the analysis with the viability dye 7-AAD (Life Technologies). When indicated, populations were cytospun (Thermo Cytospin 4) at 800 r.p.m. for 2 min, or allowed to settle at 37 °C in fully humidified chambers containing 5% CO onto poly-l-lysine-coated slides (Polysciences). The cells were subsequently fixed in 4% paraformaldehyde (pH 7.4, Electron Microscopy Sciences) before staining. Isolated crypts were counted and embedded in Matrigel (Corning 356231 growth factor reduced) at 5–10 crypts per μl and cultured in a modified form of medium as described previously13. Unless otherwise noted, Advanced DMEM (Gibco) was supplemented by EGF 40 ng ml−1 (R&D), Noggin 200 ng ml−1 (Peprotech), R-spondin 500 ng ml−1 (R&D or Sino Biological), N-acetyl-l-cysteine 1 μM (Sigma-Aldrich), N2 1X (Life Technologies), B27 1X (Life Technologies), Chiron 10 μM (Stemgent), Y-27632 dihydrochloride monohydrate 20 ng ml−1 (Sigma-Aldrich). Colonic crypts were cultured in 50% conditioned medium derived from L-WRN cells supplemented with Y-27632 dihydrochloride monohydrate 20 ng ml−1, as described43. Approximately 25–30 μl droplets of Matrigel with crypts were plated onto a flat bottom 48-well plate (Corning 3548) and allowed to solidify for 20–30 min in a 37 °C incubator. Three hundred microlitres of crypt culture medium was then overlaid onto the Matrigel, changed every 3 days, and maintained at 37 °C in fully humidified chambers containing 5% CO . Clonogenicity (colony-forming efficiency) was calculated by plating 50–300 crypts and assessing organoid formation 3–7 days or as specified after initiation of cultures. Palmitic acid (Cayman Chemical Company 10006627 conjugated to BSA), oleic acid (Sigma O1008), lipid mixture (Sigma L0288), or GW501516 (Enzo) were added immediately to cultures at 30 μM (palmitic acid, oleic acid), 2% (lipid mixture), and 1 μM (GW501516). 4-OH tamoxifen (Calbiochem, 579002, 10 nM) was added to organoid cultures derived from PpardL/L; Villin-CreERT2 (Ppard IKO) crypts to ensure Ppard excision in the ex vivo fatty acid or GW501516 experiments. Isolated ISCs or progenitor cells were centrifuged for 5 min at 250g, re-suspended in the appropriate volume of crypt culture medium (500–1,000 cells μl−1), then seeded onto 25–30 μl Matrigel (Corning 356231 growth factor reduced) containing 1 μM Jagged (Ana-Spec) in a flat bottom 48-well plate (Corning 3548). Alternatively, ISCs and Paneth cells were mixed after sorting in a 1:1 ratio, centrifuged, and then seeded onto Matrigel. The Matrigel and cells were allowed to solidify before adding 300 μl of crypt culture medium. The crypt media was changed every second or third day. Organoids were quantified on days 3, 7 and 10 of culture, unless otherwise specified. For secondary organoid assays, either individual primary organoids or many primary organoids were mechanically dissociated and then replated, or organoids were dissociated for 10 min in TrypLE Express at 32 °C, resuspended with SMEM (Life Technologies), centrifuged (5 min at 250g) and then resuspended in cold SMEM with the viability dye 7-AAD. Live cells were sorted and seeded onto Matrigel as previously described in standard crypt media (not supplemented with lipids or GW501516). Secondary organoids were enumerated on day 4, unless otherwise specified. Human biopsies were obtained from patients with informed consent undergoing intestinal resection at the Massachusetts General Hospital (MGH). The MGH Institutional Review Board committee and Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects approved the study protocols. Crypts were isolated43, embedded in Matrigel and subsequently exposed to lipid mixture, palmitic acid or GW501516 (as described in earlier). Cultures were passaged weekly and maintained for 3–4 weeks. To passage, equal numbers of organoids from each condition were disrupted with trypsin/EDTA. Numbers of organoids were counted 4–7 days after passaging into control media. Counts were normalized to numbers of organoids present in control wells and plotted. Statistical significance was calculated by performing analysis of variance (ANOVA) multiple comparisons of the means for each group. For quantitative RNA expression analysis, organoids were dissociated, cells were selected as a live population by flow cytometry (7-AAD, Life Technologies), and sorted into Tri Reagent (Life Technologies) for RNA isolation. After 5 days of culturing, intestinal organoids were placed into Karnovsky’s KII solution (2.5% glutaraldehyde, 2.0% paraformaldehyde, 0.025% calcium chloride, in a 0.1 M sodium cacodylate buffer, pH 7.4) and fixed overnight. Subsequently, they were post-fixed in 2.0% osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in graded ethanol solutions, infiltrated with propylene oxide/Epon mixtures, flat embedded in pure Epon, and polymerized overnight at 60 °C. Then 1-μm sections were cut, stained with toluidine blue, and examined by light microscopy. Representative areas were chosen for electron microscopic study and the Epon blocks were trimmed accordingly. Thin sections were cut with an LKB 8801 ultramicrotome and diamond knife, stained with Sato’s lead, and examined in a FEI Morgagni transmission electron microscope. Images were captured with an AMT (Advanced Microscopy Techiques) 2K digital CCD camera. For RNA sequencing (RNA-seq), total RNA was extracted from 200,000 sorted Lgr5-GFPhi ISCs and Lgr5-GFPlow progenitors by pooling 2–5 71-week-old HFD male or control mice using Tri Reagent (Life Technologies) according to the manufacturer’s instructions, except for an overnight isopropanol precipitation at −20 °C. From the total RNA, poly(A)+ RNA was selected using Oligo(dT) -Dynabeads (Life technologies) according to the manufacturer’s protocol. Strand-specific RNA-seq libraries were prepared using the dUTP-based, Illumina-compatible NEXTflex Directional RNA-Seq Kit (Bioo Scientific) according to the manufacturer’s directions. All libraries were sequenced with an Illumina HiSeq 2000 sequencing machine. For RNA-seq data analysis, raw stranded reads (40 nucleotides) were trimmed to remove adaptor and bases with quality scores below 20, and reads shorter than 35 nucleotides were excluded. High-quality reads were mapped to the mouse genome (mm10) with TopHat version 1.4.1 (ref. 44), using known splice junctions from Ensembl Release 70 and allowing at most two mismatches. Genes were quantified with htseq-count (with the ‘intersect strict’ mode) using Ensembl Release 70 gene models. Gene counts were normalized across all samples using estimateSizeFactors from the DESeq R/Bioconductor package45. Differential expression analysis was also performed between two samples of interest with DESeq. GSEA (http://software.broadinstitute.org/gsea/index.jsp) was performed by using the pre-ranked (according to their ratios) 8,240 differentially expressed genes as the expression data set. Motif Analysis was performed using Haystack motif enrichment tool: http://github.com/lucapinello/Haystack46. In total, 24 single Lgr5-GFPhi ISCs and 72 single Lgr5-GFPlow progenitor cells were sorted from control or HFD-fed mice (n = 2 mice per group) for single-cell gene expression analysis. For one-tube single-cell sequence-specific preamplification, individual primer sets of β-catenin target genes (total of 96, Supplementary Table 2) were pooled to a final concentration of 0.1 mM for each primer. Single cells were directly sorted into 96-well plates containing 5 μl RT–PCR master mix (2.5 μl CellsDirect reaction mix, Invitrogen; 0.5 μl primer pool; 0.1 μl reverse transcriptase/Taq enzyme, Invitrogen; 1.9 μl nuclease-free water) in each well. Immediately after, plates were placed on PCR machine for preamplification. Sequence-specific preamplification PCR protocol was as following: 60 min at 50 °C for cell lysis and sequence-specific reverse transcription; then 3 min at 95 °C for reverse transcriptase inactivation and Taq polymerase activation. cDNA was then amplified by 20 cycles of 15 s at 95 °C for initial denaturation, 15 min at 60 °C for annealing and elongation. After preamplificiation, samples were diluted 1:5 before high-throughput microfluidic real-time PCR analysis using Fluidigm platform. Amplified single-cell cDNA samples were assayed for gene expression using individual qRT–PCR primers and 96.96 dynamic arrays on a BioMark System by following manufacturers protocol (Fluidigm). To confirm PPAR-δ-mediated induction of the most upregulated genes (n = 3 mice, 24 ISCs and 72 progenitors per group), or for single-cell analysis of organoid composition (n = 3 mice, 48 cells per group) and db/db mice (n = 3, 48 cells per group) standard single-cell qRT–PCR was performed using preamplified cDNA with corresponding primers. For Fluidigm analysis, threshold cycle (C ) values were calculated using the BioMark Real-Time PCR Analysis software (Fluidigm). See Supplementary Information for raw gene expression data. Gene expression levels were estimated by subtracting the C values from the background level of 35, which approximately represent the log gene expression levels. The t-Distributed stochastic neighbour embedding (t-SNE) analysis47 was performed using the MATLAB toolbox for dimensionality reduction. Differential expression analysis was conducted using the two-sided Wilcoxon–Mann–Whitney rank sum test implemented in the R coin package (https://www.r-project.org). P values were adjusted for multiple testing48 using the p.adjust function in R with method = ‘fdr’ option. Fold changes were calculated as the difference of median of log expression levels for the two cell populations. Split violin plots were generated using the vioplot package and the vioplot2 function in R (https://gist.github.com/mbjoseph/5852613). The heatmap for β-catenin target genes was generated with the MultiExperiment Viewer (MeV) program (http://www.tm4.org/mev.html) using the correlation-based distance and average linkage method as parameters of the unsupervised hierarchical clustering of genes. The heatmap for organoid composition was generated using MATLAB. The percentages of Jag1/Jag2-upregulated cells were calculated based on the number of single cells whose log expression was above 15. Approximately 25,000 cells were sorted into Tri Reagent (Life Technologies) and total RNA was isolated according to the manufacturer’s instructions with following modification: the aqueous phase containing total RNA was purified using the RNeasy plus kit (Qiagen). RNA was converted to cDNA with the cDNA synthesis kit (Bio-Rad). qRT–PCR was performed with diluted cDNA (1:5) in three wells for each primer and SYBR green master mix (Bio-Rad) on Bio-Rad iCycler RT–PCR detection system. For organoid experiments, 1,000 live cells were sorted and qRT–PCR optimized for low cell numbers (<1,000) was performed after sequence specific pre-amplification (cDNA diluted 1:200 in three wells for each primer) as described in single-cell gene expression analysis. All qRT–PCR experiments were repeated at least three independent times. Primers used are listed on Supplementary Table 1. ApcL/L; Lgr5-EGFP-IRES-CreERT2 mice were treated with vehicle or GW501516 for 1 month, and then injected with two intraperitoneal doses of tamoxifen. Four days later, Apc-null Lgr5-GFPhi ISCs and Lgr5-GFPlow progenitors were sorted by flow cytometry, as described earlier. For primary cell transplantations, 10,000 Apc-null Lgr5-GFPhi ISCs and Lgr5-GFPlow progenitors were resuspended into 90% crypt culture media (as described) and 10% Matrigel, then transplanted into the colonic lamina propria of C57BL/6 recipient mice by optical colonoscopy using a custom injection needle (Hamilton Inc., 33-gauge, small Hub RN NDL, 16 inches long, point 4, 45 degree bevel, like part number 7803-05), syringe (Hamilton Inc. part number 7656-01), and transfer needle (Hamilton Inc. part number 7770-02). Optical colonoscopy was performed using a Karl Storz Image 1 HD Camera System, Image 1 HUB CCU, 175 Watt Xenon Light Source, and Richard Wolf 1.9mm/9.5 Fr Integrated Telescope (part number 8626.431). Four injections were performed per mouse. Mice then underwent colonoscopy 8 weeks later to assess tumour formation. Colonoscopy videos and images were saved for offline analysis. Following sacrifice, the distal colons were excised and fixed in 10% formalin, then examined by haematoxylin and eosin section to identify adenomas. Histology images were reviewed by gastrointestinal pathologists who were blinded to the treatment groups (S.S., V.D. and Ö.H.Y.). All experiments reported in Figs 1, 2, 3, 4, 5 were repeated at least three independent times, except for Figs 3a, 4c, d, which were repeated twice. All samples represent biological replicates. For mouse organoid assays, 2–4 wells per group with at least 3 different mice were analysed. For human organoid assays, 4 wells per group with 4 different patient samples were analysed and experiments were repeated 4 times. All centre values shown in graphs refer to the mean. For statistical significance of the differences between the means of two groups, we used two-tailed Student’s t-tests. Statistical significance in Fig. 3k was calculated by performing ANOVA multiple comparisons of the means for each group. No samples or animals were excluded from analysis, and sample size estimates were not used. Animals were randomly assigned to groups. Studies were not conducted blinded, with the exception of all histological analyses and Fig. 5c, h. All experiments involving mice were carried out with approval from the Committee for Animal Care at MIT and under supervision of the Department of Comparative Medicine at MIT.


News Article | October 5, 2016
Site: www.nature.com

The ExbB construct with and without a C-terminal 6×His tag was subcloned into pET26b (Novagen). ExbD was subcloned into pACYCDuet-1 vector (Novagen) with an N-terminal Strep-tag and a C-terminal 10×His tag. ExbD was also subcloned into a pCDF-1b vector (Novagen) containing a C-terminal TEV protease site followed by a 10×His tag. An ExbD construct containing a C-terminal TEV protease site (preceded by a Gly-Gly-Gly linker for efficient digestion by TEV protease) followed by a 10×His tag was constructed by deletion of the sequence encoding the periplasmic domain of ExbD (residues 50–141). TonB was cloned into a pACYCDUET-1 vector with an N-terminal 10×His tag followed by a TEV protease site. Mutants of TonB (C18A), ExbD (D25A, N78C and E113C), and ExbB (C25S) were prepared by site-directed mutagenesis (primer sequences for all cloning and mutagenesis experiments are available upon request). The sequences of all plasmid constructs and mutations were verified by sequence analysis (Macrogen USA and Eurofins Genomics GmbH). Expression of ExbB with a C-terminal 6×His tag was performed by transforming E. coli BL21(DE3) cells (NEB) with the pET26b/ExbB vector. Co-expression was performed by co-transforming E. coli BL21(DE3) cells with the respective ExbB, ExbD, and/or TonB plasmids. For all transformations, cells were plated onto LB agar plates supplemented with appropriate antibiotics. Colonies were then used for a starter culture to inoculate 12 flasks containing either 1 l 2×YT medium (Ton subcomplex) or SelenoMet medium supplemented with l-methionine at 40 mg/l (Molecular Dimensions) (Ton complex), with appropriate antibiotics. Cultures were grown at 37 °C with shaking at 220 r.p.m. until they reached an OD of 0.5–1.0, induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) to 0.1 mM final concentration, and then allowed to continue to grow overnight at 28 °C. For selenomethionine-substituted samples for experimental phasing, B834(DE3) cells (NEB) were co-transformed with pET26b/ExbB and pCDF-1b/ExbD plasmids. Single colonies were used to inoculate 12 flasks containing 1 l SelenoMet medium (Molecular Dimensions) supplemented with 40 mg/ml l-selenomethionine and appropriate antibiotics. Cultures were grown at 37 °C with shaking at 220 r.p.m. until they reached an OD of 0.5–1.0, induced with IPTG to 0.1 mM final concentration, and then allowed to continue to grow overnight at 28 °C. Cells were harvested and used immediately or stored at −80 °C. For purification, cells were resuspended in either 1×PBS (Ton subcomplex) or TBS (Ton complex) supplemented with 100 μM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 100 μM DNase, and 50 μg/ml lysozyme, and disrupted with two passages through an EmulsiFlex-C3 (Avestin) operating at ~15,000 p.s.i. Membranes were pelleted by ultracentrifugation in a Type 45 Ti Beckman rotor at 200,000g for 1 h at 4 °C. Membranes were then resuspended in 1×PBS or TBS using a dounce homogenizer and solubilized by the addition of Triton X-100 (Ton subcomplex) or DDM (Anatrace) (Ton complex) to a final concentration of 1% by stirring at medium speed for 1 h to overnight at 4 °C. Insoluble material was pelleted by ultracentrifugation in a Type 45 Ti Beckman rotor at 200,000g for 1 h at 4 °C and the supernatant was used immediately. Immobilized metal affinity chromatography (IMAC) was performed on an AkTA Purifier (GE Healthcare) using a 15-ml Ni-NTA agarose column (Qiagen) equilibrated with 1×PBS or TBS supplemented with 0.1% Triton X-100 or 0.1% DDM. The supernatant was supplemented with 10 mM imidazole and loaded onto the column. The column was washed in three steps with 1×PBS or TBS supplemented with 20, 40 and 60 mM imidazole, respectively, and eluted with 1×PBS or TBS supplemented with 250 mM imidazole in 2-ml fractions. Fractions were analysed by SDS–PAGE and those fractions containing the complex were pooled. To remove the 10×His tag, TEV protease was added to the sample at 0.1 mg/ml final concentration and rocked overnight at 4 °C. For the Ton complex, the sample was then diluted 2–3 times with 25 mM HEPES, pH 7.3, and 0.1% DDM and loaded onto an anion exchange 6-ml ResourceQ column (GE Healthcare). Elution was performed with a 0–1 M NaCl gradient over 5 column volumes. For the Ton subcomplex, the sample was concentrated using an Amicon Ultra-15 Centrifugal Filter Unit with a 50-kDa MW cut-off (Millipore), filtered, and purified by size-exclusion chromatography using a Superdex 200 HL 16/600 column (GE Healthcare) at a flow rate of 0.5–1.0 ml/min. The buffer consisted of 20 mM HEPES-NaOH, pH 7.0, 150 mM NaCl, 0.01% NaN , and 0.08% C E . For the Ton complex, eluted fractions were concentrated using an Amicon Ultra-15 Centrifugal Filter Unit with a 100-kDa MW cut-off (Millipore), and passed over a Superose6HR 10/30 column (GE Healthcare) at a flow rate of 0.5 ml/min using 20 mM HEPES-NaOH, pH 7.0, 150 mM NaCl, and 0.05% DDM. Far-UV circular dichroism (CD) spectra (185–260 nm) were measured in 0.1 M NaP , pH 7.0, and 0.03% DDM using quartz cuvettes with a 0.02–0.2 mm optical path length. The results were analysed using the DichroWeb package of programs42 and different sets of reference proteins, including the SMP180 set of membrane proteins. The analysis of the thermal stability of the complexes reconstituted into liposomes was measured by the temperature dependence of the CD signal amplitude at 222 nm. Thermal melting was performed in a magnetically stirred 1-cm quartz cuvette containing 10 mM HEPES, pH 7.0, and 100 mM NaCl with a rate of temperature increase of 0.5 °C/min. Melting curves were normalized to the measured value of the molar ellipticity change at 10 °C. For crystallization, samples were concentrated to ~10 mg/ml and sparse matrix screening was performed using a TTP Labtech Mosquito crystallization robot using hanging drop vapour diffusion and plates incubated at 15–21 °C. Initially, many lead conditions were observed to produce crystals with hexagonal morphology; however, none diffracted to better than ~7 Å and most suffered from anisotropy. To avoid this packing, we performed reductive methylation of our samples before crystallization using the Reductive Alkylation Kit (Hampton Research), followed by an additional size-exclusion chromatography step. This led to a condition which produced diffraction spots to ~4 Å resolution. Further optimization and screening allowed us to grow crystals in 100 mM Na-acetate, pH 4.5, 100 mM MgCl , and 25% PEG 400 that routinely diffracted to ~3.5 Å resolution or better. For heavy atom soaking, crystals were transferred to a drop containing 1 mM HgCl and incubated overnight at room temperature and then harvested directly from the soaking condition. The best native crystals for the ExbB–ExbD complex, however, were grown from 100 mM HEPES-NaOH, pH 7.0, 100 mM CaCl , and 22% PEG MME 550 and diffracted to 2.6 Å resolution; these crystals were also used for heavy atom soaking experiments. Unfortunately, none of the heavy atom soaked crystals (nor the selenomethionine substituted crystals) were useful for phasing owing to crystal pathologies, which we suspected were twinning related. However, selenomethionine substituted crystals of the ExbB –ExbD complex were obtained using 100 mM MES/imidazole, pH 6.5, 30 mM MgCl , 30 mM CaCl , 50% ethylene glycol, and 8% PEG 8000 and diffracted to 5.2 Å resolution with no twinning-related issues. Both native and selenomethionine-substituted crystals were harvested directly from the crystallization drops. Screening for diffraction quality was performed at the GM/CA-CAT and SER-CAT beamlines at the Advanced Photon Source at Argonne National Laboratory and at beamlines 5.0.1 and 8.2.1 at the Advanced Light Source at Lawrence Berkeley National Laboratory. Final datasets were collected at the SER-CAT beamline and all data were processed using either HKL200043 or Xia244. A summary of the data collection statistics can be found in Supplementary Table 1. The presence of both components of the Ton subcomplex within the crystals was confirmed by SDS–PAGE and mass spectrometry analyses of harvested crystals. For phasing the ExbB–ExbD complex structure, three datasets were collected on selenomethionine substituted crystals of the ExbB –ExbD complex at a wavelength of 0.979 Å. The data were processed with Xia244 and, based on non-isomorphism, one dataset was removed. The final two datasets were processed together in space group P4 2 2 to a final resolution of 5.2 Å. Selenium sites (35 total) were located using HKL2MAP45 after 5,000 tries within SHELXD at a resolution range of 20–6 Å. The sites were then fed into AutoSol (PHENIX)46 which removed one site, producing a phase-extended density-modified electron density map into which we could build an initial poly-alanine model. Five-fold symmetry was clearly observed, with each monomer consisting of very elongated α-helices, and directionality was determined on the basis of the predicted topology of ExbB, which contains a single large cytoplasmic domain. This model was then used as a search model to solve the native and Hg-soaked structures by molecular replacement using PHASER/PHENIX46, 47 and the sequence docked on the basis of anomalous peaks from the SeSAD dataset. The ExbB–ExbD complex was solved in space group P2 to 2.6 Å resolution with R/R values of 0.21/0.26 and the Hg-soaked structure in space group P2 2 2 to 3.5 Å resolution with R/R values of 0.25/0.30. All model building was performed using COOT and subsequent refinement done in PHENIX46. r.m.s.d. analysis was performed within PyMOL (Schrödinger). Electrostatic surface properties (calculated using the Linearized Poisson-Boltzman Equation mode with a solvent radius of 1.4), including generation of the electric field lines, were analysed and visualized using the APBS plugin within PyMOL (Schrödinger). Buried surface area was calculated using the PDBePISA server48. Structure-related figures were made with PyMOL (Schrödinger) and Chimera49 and annotated and finalized with Adobe Photoshop and Illustrator. Coordinates and structure factors for the ExbB/ExbD complexes have been deposited into the Protein Data Bank (PDB accession codes 5SV0 and 5SV1). For 2D crystallization experiments, the Ton subcomplex (ExbB–ExbD) was extracted and purified by IMAC as previously described. The sample was passed over a Superose 12 HR 10/30 column using 20 mM Tris-HCl, pH 7, 150 mM NaCl, 0.01% NaN , and 0.035% Triton X-100. The purified complex was then mixed with a solution stock of E. coli polar lipid (Avanti Polar Lipids, Inc.) at 10 mg/ml in 2% Triton X-100, to reach final concentrations of 0.5–1.0 mg/ml protein and 0.1–0.4 mg/ml lipid. The lipid-protein-detergent samples solutions were placed into Mini Slide-A-Lyser dialysis devices (Pierce) with a 20-kDa MW cutoff, and dialysed in 1 l of 25 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 0.01% NaN at 4 °C. Aliquots of dialysed samples were observed periodically by electron microscopy to monitor the formation of 2D crystals. Sample preparation for electron microscopy was carried out by applying a 5-μl drop of protein-lipid material on a glow discharged carbon-coated electron microscopy grid. Staining was performed by addition of 1% (w/v) uranyl acetate and incubation for 1 min. Grids were then imaged on a Tecnai G2 200 LaB6 electron microscope operating at 200 kV at the Institut de Microbiologie de la Méditerranée. Images were recorded with a 2K Eagle CCD camera. The best 2D crystals were selected through observation of the power spectrum of the images using ImageJ software41. Selected images were processed using the IPLT Correlation Averaging suite program50. A filtered image was generated by optical filtering of the low resolution spots, and padded to contain only 4–6 unit cells. The padded image was cross-correlated with the original large image. The positions of the cross-correlation peaks were determined and used to extract sub-images that were summed to generate an average image of the 2D unit cell. Site-directed spin labelling was used to covalently attach the spin label (1-oxyl-2,2,5,5-tetramethyl-∆3-pyrroline-3-methyl) methanethiosulfonate (MTSL) (Toronto Research Chemicals) to Cys25 on ExbB and to cysteines engineered at positions 78 and 113 on ExbD (N78C, E113C; ExbD constructs were in the pACYC vector containing an N-terminal strep-tag and a C-terminal 10×His tag for the Ton subcomplex, and in the pCDF-1b vector for the Ton complex). For labelling with MTSL, samples were first incubated with 2–10 mM dithiothreitol (DTT) for 1–2 h and the DTT then removed by passage over a HiTrap desalting column (GE Healthcare) or during anion exchange (Ton complex). Samples were then incubated with a 10× molar excess of MTSL overnight at 4 °C and then passed over a Superose 6HR 10/30 gel filtration column (GE Healthcare) using 20 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 0.08% C E or 0.03% DDM (Ton subcomplex); or 20 mM HEPES-NaOH, pH 7.0, 150 mM NaCl, and 0.05% DDM (Ton complex). For DEER measurements, the samples were diluted with D O to a final concentration of 30% and cryoprotected with 10% v/v D8-glycerol before being flash frozen in liquid nitrogen. Continuous wave (CW) electron paramagnetic resonance (EPR) experiments were carried out at room temperature on a bench-top X-band MiniScope MS 400 (Magnettech by Freiberg Instrument) at 9.5 GHz (X-band) with 2.5 mW microwave power, 15 mT sweep width and 0.15 mT modulation amplitude. Spin labelling efficiency was calculated from the second integral of the derivative spectra compared to a standard spin concentration of 100 μM (Tempol in water). The ExbB native cysteine C25 was labelled with a 50% efficiency, while the ExbD mutants were labelled with efficiencies >80%. DEER measurements were initially performed at ETH Zurich on a commercial Bruker ELEXSYS-II E580 Q-band spectrometer (34–35 GHz) and later on a Bruker ELEXSYS E580Q-AWG dedicated pulse Q-band spectrometer operating at 34–35 GHz. Both spectrometers were equipped with a TWT amplifier (150 W) and a home-made rectangular resonator (from ETH Zurich) enabling the insertion of 30–40 μl sample volume in quartz tubes with 3 mm outer diameter51. Dipolar time evolution data were acquired using the four-pulse DEER experiment at 50 K. All pulses were set to be rectangular with 12 ns length, with the pump frequency at the maximum of the echo-detected field swept spectrum, 100 MHz higher than the observer frequency. Deuterium nuclear modulations were averaged by increasing the first interpulse delay by 16 ns for 8 steps as previously described51. The background of the normalized DEER primary data (V(t)/V(0)) was fitted with optimized dimensions from 2.5 to 3.2 and the resulting normalized secondary data (F(t)/F(0)) were converted by model-free Tikhonov regularization to distance distributions with the software DeerAnalysis201552, 53. The simulation of the possible spin label rotamers populated at selected positions in the protein was performed using the Matlab program package MMM2015.1 using the MTSL ambient temperature library54. The ExbB –ExbD complex (ExbD was in the pACYC vector containing an N-terminal strep-tag and a C-terminal 6×HIS tag) was expressed and purified as described earlier. To prepare the sample for crosslinking, the sample was incubated at 4 °C with 5 mM DTT for at least 1 h. The DTT was then removed using a desalting column in 20 mM HEPES, pH 7.0, 150 mM NaCl, and 0.1% DDM. The crosslinker 1,8-bismaleimidodiethylenglycol (BM(PEG) ) (Pierce) was added at a final concentration of 0.2 mM and the reaction was incubated at 4 °C overnight. The sample was concentrated and passed over a Superose 6HR 10/30 gel filtration column using 20 mM HEPES-NaOH, pH 7.0, 150 mM NaCl, and 0.035% DDM on an AkTA Purifier system (GE Healthcare). The results were visualized by SDS–PAGE analysis. Protein complexes were reconstituted into liposomes by dialysis of the protein–lipid–detergent mixture. Lipids (DOPG, DOPC and DOPE) dissolved in chloroform were mixed in a molar ratio of 2:3:5. Chloroform was removed by vortexing in a stream of nitrogen gas in a glass tube followed by drying in vacuum for 2–3 h. The lipid film was hydrated in 1 ml TN buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl), followed by five cycles of freeze–thaw and sonication using a water bath sonicator until the suspension of lipids became clear (10–15 min). For proteoliposome preparation, small unilamellar vesicles (SUVs) were mixed with octylglucoside (final concentration, 2%) and then proteins added to achieve a molar ratio of total lipid to protein ∼500–2,000 mol/mol. After 1 h incubation in ice, the lipid–protein–detergent mixture was dialysed into 10 mM Tris-HCl, pH 7.5, 0.3 M sucrose, and 50 mM KCl for 30–40 h using a dialysis membrane with a MW cut-off pore size of 10 kDa. Mueller-Rudin type planar bilayer membranes were formed on a 0.2-mm diameter aperture in a partition that separates two 1-ml compartments, using a mixture of lipids, DOPG, DOPC and DOPE, at a molar ratio of 2:3:5 (10 mg/ml) in n-decane, applied by a brush technique55. The aqueous solution in both compartments consisted of 2 mM KP , pH 7.0, and 0.1 M and 0.4 M KCl in the cis- and trans-compartments, respectively. To study the pH dependence of channel activity, bathing solutions were buffered with 2 mM Na-acetate (pK 4.8), Na-cacodylate (pK 6.2), and Tris (pK 8.3). The pH of the bathing solution was changed by adding 10–20 μl 0.1 M HCl or KOH. The cis-side of the planar bilayer is defined as that to which the electrical potential is applied. Proteoliposomes, 0.1–2 μl, were added to the trans-compartment, and the solutions were stirred until the transmembrane current appeared. A large concentration of an osmolyte inside of the liposomes and the transmembrane KCl concentration gradient caused proteoliposome fusion with the pre-formed planar lipid membrane bilayer. The transmembrane current was measured in voltage-clamp mode with Ag/AgCl electrodes and agar bridges, using a BC-525C amplifier (Warner Instruments). The single-channel conductance of the ExbB–ExbD complexes was measured in symmetrical salt conditions: 0.1 M KCl solution, pH 7.5, at a holding potential of +50 or −50 mV. For ion selectivity experiments, zero-current potential (V ) was determined from volt-ampere characteristics measured in asymmetric salt conditions. Relative cation/anion permeability was calculated using the Goldman-Hodgkin-Katz equation56.


Larson S.B.,University of California at Irvine | Day J.S.,University of California at Irvine | Nguyen C.,Hampton Research | Cudney R.,Hampton Research | McPherson A.,University of California at Irvine
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2010

Bovine pancreatic ribonuclease A (RNase A) was crystallized from a mixture of small molecules containing basic fuchsin, tobramycin and uridine 5′-monophos-phate (U5P). Solution of the crystal structure revealed that the enzyme was selectively bound to U5P, with the pyrimidine ring of U5P residing in the pyrimidine-binding site at Thr45. The structure was refined to an R factor of 0.197 and an Rfree of 0.253. © 2010 International Union of Crystallography All rights reserved.


McPherson A.,University of California at Irvine | Cudney B.,Hampton Research
Acta Crystallographica Section F:Structural Biology Communications | Year: 2014

For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth. The improvement process is referred to as optimization, and it entails sequential, incremental changes in the chemical parameters that influence crystallization, such as pH, ionic strength and precipitant concentration, as well as physical parameters such as temperature, sample volume and overall methodology. It also includes the application of some unique procedures and approaches, and the addition of novel components such as detergents, ligands or other small molecules that may enhance nucleation or crystal development. Here, an attempt is made to provide guidance on how optimization might best be applied to crystal-growth problems, and what parameters and factors might most profitably be explored to accelerate and achieve success. © 2014 International Union of Crystallography.


Larson S.B.,University of California at Irvine | Day J.S.,University of California at Irvine | Nguyen C.,Hampton Research | Cudney R.,Hampton Research | McPherson A.,University of California at Irvine
Acta Crystallographica Section D: Biological Crystallography | Year: 2010

Human methemoglobin was crystallized in a unique unit cell and its structure was solved by molecular replacement. The hexagonal unit cell has unit-cell parameters a = b = 54.6, c = 677.4 Å, with symmetry consistent with space group P6122. The unit cell has the second highest aspect ratio of all unit cells contained in the PDB. The 12 molecules in the unit cell describe a right-handed helical filament having no polarity, which is different from the filament composed of HbS fibers, which is the only other well characterized fiber of human hemoglobin. The filaments reported here can be related to canonical sickle-cell hemoglobin filaments and to an alternative sickle-cell filament deduced from fiber diffraction by slight modifications of intermolecular contacts. © 2010 International Union of Crystallography.


McPherson A.,University of California at Irvine | Nguyen C.,Hampton Research | Cudney R.,Hampton Research | Larson S.B.,University of California at Irvine
Crystal Growth and Design | Year: 2011

An alternative approach to promoting the crystallization of proteins is to enhance intermolecular contacts between macromolecules or to eliminate intermolecular interactions or interactions with solvent that might inhibit crystallization. Site-specific mutations have been employed, as have truncations by genetic or proteolytic means. There are, however, significant problems. Because the structure of the target macromolecule is unknown, there may be no good basis for the design of mutants or truncations. In addition, the approach requires that the protein be produced by recombinant DNA technology, which is frequently not the case. We have attempted to address these issues by initiating experiments based on two ideas. The first is that a wide variety of conventional small molecules might be systematically introduced into mother liquors during crystallization screening. By incorporation into the crystal lattice, the additional intermolecular interactions that the small molecules provide might enhance crystal nucleation and growth. A second approach that we are pursuing is the chemical modification of various amino acid side chains using traditional protein chemistry. We believe that in some cases chemically modified proteins might be induced to crystallize or crystallize better than the native protein. © 2011 American Chemical Society.


PubMed | University of California at Irvine and Hampton Research
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2014

For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth. The improvement process is referred to as optimization, and it entails sequential, incremental changes in the chemical parameters that influence crystallization, such as pH, ionic strength and precipitant concentration, as well as physical parameters such as temperature, sample volume and overall methodology. It also includes the application of some unique procedures and approaches, and the addition of novel components such as detergents, ligands or other small molecules that may enhance nucleation or crystal development. Here, an attempt is made to provide guidance on how optimization might best be applied to crystal-growth problems, and what parameters and factors might most profitably be explored to accelerate and achieve success.

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