EMBO/sd-character-level-ner huggingface dataset:
Figure 3A. Schematic showing the primers used to make the PCR product from the library plasmid B. Schematic of 'platforms' integrated into the intS and the galK locus of the E. coli chromosome. These platforms consist of the sequence for a fluorescent protein (yfp or cfp), a functioning resistance marker (chlR or kanR), and a truncated and thus defunct resistance marker (kanR or ampR). A PCR product shown in A was introduced by recombineering (black crossed lines) into the platforms. C. Schematic of final reporter constructs in the chromosome. The functional resistance marker is replaced by the promoter of interest, and the defunct resistance marker is completed, yielding promoters driving yfp and cfp, respectively. D. Response times of the LlacO-1 promoter (Lutz & Bujard, 1997), induced with 1 mM IPTG at time zero, measured with YFP and CFP constructs in the same cell (r = 0.81 ± 0.04, p = 6 · 10-12).
Suppression of Wnt signaling in intestinal basal crypts and ISPCs of aging telomerase‐deficient micemRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 5 mice per group).Figure 2A mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 5 mice per group). B Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group). C mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).
Figure 8Growth of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1 is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL SorA and incubated at 30 oC for 3 d.
Figure 5A, Control and degron ORP2 cells were co-plated, and after 1 day incubation in 5 % LPDS, cells were loaded with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for the indicated times. Intracellular cholesterol was labeled with D4H and imaged by widefield epifluorescence microscopy. Asterisk indicates region of ORP2-depleted cells. CM, complete medium. Dashed lines separate ORP2 expressing and depleted cells. B, Quantification of A. Mean ± SD, n = 15-27 cells pooled from 2 independent experiments. Student's t-test. C, E, Control and degron ORP2 cells were co-plated, and after 1 day in 5 % LPDS, treated with 50 μg/ml LDL and IAA for the indicated times. Asterisk indicates an ORP2-depleted cell. Double labeling of intracellular D4H and Lamp1 (C) or intracellular D4H and internalized integrin β1 (E). Images were acquired by confocal microscopy. Red box indicates a peripheral region toward the leading edge in the control cell, and blue box a corresponding region in the ORP2-depleted cell. D, F, Fraction of D4H area overlapping with lamp1 (D) and D4H area positive for internalized integrin β1 (F) in a peripheral region toward the leading edge (exemplary box as inset). Mean ± SD, n = 20-30 cells pooled from 2 independent experiments. Student's t-test.
Figure 7A. Accumulation of the HY5 protein in WT and phyB1B2 (phyB) plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein. B. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room. C. The relative FCR activities in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room. D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.
Benchmarking retinal cells using the human neural retina atlas.Figure 6A) Correlation analysis of scRNA-seq data of hiPSC-derived cone photoreceptors (week 15) against fetal cone photoreceptors (Welby et al, 2017), as well as adult cone and rod photoreceptors from this human neural retina atlas. B) Principal component analysis to assess transcriptome similarity of hiPSC-derived cone photoreceptors to fetal and adult photoreceptors. C) t-SNE analysis of the human Müller glia cell line MIO-M1 with the retinal cell types identified in this human neural retina atlas. D) Correlation analysis of MIO-M1 with all major human retinal cell types. E) Top ranked differentially expressed genes identified in MIO-M1 compared to other retinal cell types based on logistic regression score.
Figure 7(A) Extracts of DIV 7 hippocampal neurons electroporated with pSuper control or with three different DCLK1 shRNAs at DIV0. Samples were analyzed by western blot with indicated antibodies. (B, E) Representative images of a hippocampal cell transfected at DIV10 with RFP (to visualize transfected cells), pSUPER (control) or DCLK1 shRNA, fixed at DIV14 and stained for EB3. Black lines indicate the neuronal soma outline. Scale bars = 10 µm. (C, D) Quantification of the number of EB3 comets in the dendrite (C) and in the soma (D). 25-26 neurons were analyzed per condition, N=2. Error bars indicate SEM, *** - p < 0.001, ** - p<0,01 (unpaired t-test). (F-I) Hippocampal neurons were co-transfected with MACF18-GFP and pSUPER (control) or DCLK1 shRNA and the dynamics of microtubules in dendrites was traced using life-cell microscopy. (F) Representative kymographs of dynamic microtubules ends growth in dendrites with and without photoablation. Scale bar = 10 µm (C). (G, I) Quantification of the fraction of MACF18 events moving retrogradely and anterogradely in a dendrite before (G) and after (I) photoablation. Eighteen neurons were analyzed per condition; N=2. (H) Scheme of live-cell imaging after photoablation. (J, K) Biotin pull-downs (PD) from extracts of HEK293 transfected with biotin-tagged DCLK1 and (J) KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB, KIF5B-MDC-HA-FRB constructs and probed for HA/DCLK1, (K) different GFP-tagged KIF1A truncation constructs and probed for GFP/DCLK1. The ratio input/pellet is 2% for all pull-down experiments. (L) GFP pull-down from extracts of HEK293 transfected with DCLK1 fragments and KIF1A-MDC-HA-FRB, KIF1C-MDC-HA-FRB and KIF1C-MD-HA and probed for HA/GFP. LacZ-HA was used as a negative control.
Fig.Figure 6 A. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry plus-end marker before (dark grey) and after (light grey) treatment with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin) and auxin (0.1 µM NAA). MT plus-end growth rates were monitored for 10 min before and for 10 min after each treatment. The number of MT plus-end events (EB3-mCherry) tracked per cell was 100-200/min. Mean ± s.d., (*** P < 0.001 by Student's t-test, n = 1-3 cells per condition in 4 independent replicate).C-E. Immunostaining of α-tubulin (green) in leukocytes mounted in media containing DAPI (blue, nuclear marker) and quantification of immunolabeled MTs. Cells were not or treated with 300 nM nocodazole (N) for 10 min (C). Leukocytes pretreated for 30 min with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin), adenine (10 µM) or auxin (10 µM NAA) prior to a 10-min treatment with 300 nM nocodazol (D). Scale bar 50 µm (C, D). E. Relative fluorescence intensity of MTs in leukocytes treated with nocodazole, or nocodazole and hormones as described for (D) compared to untreated (black bar) cells. Mean ± s.d., (P < 0.05 by two-way Anova, n = 18-33 cells per condition in 3 independent replicates).
Figure 6(A) Example trace of PP7-GAL10 transcription in 2% galactose. (B) Boxplot of the onset of the first transcription event in different doses of galactose. The box indicates quartiles, the horizontal line inside the box indicates the median, the whiskers indicates 1.5 times the interquartile range of the box. Data points outside the whiskers are indicated with circles (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells). (C) The autocorrelation was used to interpret the burst duration and burst frequency changes. Burst duration is measured by the intersect of the two linear fits. When burst duration is constant, the amplitude is inversely related to the burst frequency. (D) Burst duration from autocorrelation is constant across 4 different galactose concentrations (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. (E) Amplitude of autocorrelation decreases (burst frequency increases) at higher doses of galactose. (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. (F) Example traces of two single cells exposed to two different doses of galactose. (G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM.
Figure 5DNA and crRNA sequence used for experiments with tandem target-multiple PAM constructs. DNA sequence is shown for the symmetric construct with each target having only one adjacent PAM and schematic representation is shown for multiple-PAM adjacent to the second target containing constructs. Histogram showing the percentage of binding events starting at either target. The values are averages of five measurements over three different days. Error bars represent standard error of the mean. DNA and crRNA sequence used for experiments with a full target and multiple PAMs. DNA sequence is shown for the single-target single-PAM construct and schematic representation is shown for multiple-PAM containing constructs. An example trace showing the addition of Cy3-labeled DNA target and the first binding event. Δτon is indicated. Scatter plot showing the on-target binding rate (kon-target) for each construct. Binding rate decreases moderately with increasing number of adjacent PAMs. The values are averages of four measurements over three days. Error bars represent standard error of the mean. kon-target was calculated using the equation kon-target=1/ (CxΔτon) where C is the molarity of DNA target in the channel.
Figure 6(A) Representative images of intestines from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) were measured in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi. (B) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (C) Intestine tissues from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group) were collected for immunohistochemistry staining. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm. (D) Immunofluorescence staining of intestine tissues from Srcapfl/fl; Lgr5GFP-CreERT2 and Restfl/fl; Lgr5GFP-CreERT2 mice. Scale bar, 50 μm. (E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel). (F) Rest+/+ and Rest-/- mice were treated with 10 Gy's radiation and sacrificed at the indicated time points. Intestinal tissues were collected for H&E staining. Scale bars, 100 μm. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). (G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.
Whole blood transcriptome analysis in symptomatic and asymptomatic COVID-19 patients highlights distinctive immune signatures during the acute phase of infection.Figure 1A. RNA-seq of whole blood from symptomatic (n=26; median 8 days PIO) and asymptomatic (n=30; median 3 days post-hospital admission) COVID-19 patients at the acute phase of infection (SARS-CoV-2 PCR-positive) was performed. Only samples with RNA integrity number >6 were sent for sequencing and included in the analysis. Volcano plot indicating DEGs between asymptomatic and symptomatic COVID-19 patients, with thresholds of FDR < 0.05 and |FC| > 2. Numbers of over-expressed and under-expressed genes are indicated. B. PCA of symptomatic and asymptomatic COVID-19 patients based on DEGs, with FDR < 0.05 and |FC| > 2. C. Pie chart showing distribution of the functionally grouped GO terms using ClueGO. Illustrated genes were under-expressed or over-expressed in asymptomatic patients compared to symptomatic patients. Each GO term is statistically significant (Benjamini-Hochberg correction < 0.05), with a cut-off set at P < 0.05.
Figure 7Localization of Venus-Rod during unperturbed mitosis in control depleted or Bub1 depleted cells. Primary structure of Bub1 with sequence of CD1 shown and residues mutated in red. Schematic of fusion constructs used in panels (E and F). HeLa cells were depleted of Bub1 using RNAi and complemented with indicated Venus-Bub1 constructs. Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions. HeLa cells were depleted of Bub1 using RNAi Immunofluorescence images were shown for Bub1 and ZW10 localization. Quantification of Bub1 and ZW10 kinetochore levels normalized to CREST in the indicated conditions. with the indicated Bub1 fusion proteins
A small molecule IRE1α inhibitor interferes with prostate cancer cell growth in vivo.Figure 6LNCaP xenografts were grown in nude mice until palpable. Mice were then intraperitoneally injected with 0.5 mg/kg toyocamycin or saline (Vehicle) (tumor numbers:n = 15, orn = 10, respectively) twice weekly.Tumor sizes were measured weekly with calipers. Error bars indicate SEM. *P = 0.04; **P = 0.0004; ***P = 0.002 using unpaired Student's t-test.Same procedure was repeated on VCaP xenografts, with similar findings as in LNCaP xenografts (tumor numbers: n = 9, n = 11). Error bars indicate SEM. *P = 0.02 and **P = 0.0006 using unpaired Student's t-test. XBP-1S mRNA levels in LNCaP xenografts from animals treated with toyocamycin or vehicle. Tumors were harvested, RNA-extracted, and qPCR performed. Error bars indicate SEM. *P = 0.04 for LNCaP and **P = 0.02 for VCaP using unpaired Student's t-test. PCNA staining in tumors from animals treated with either toyocamycin or saline. Scale bar: 100 μm.
Chronic dose-escalated IMT1 treatment confers resistance to RKO cells through an upregulation of mitochondrial function.Figure 4A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments. B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control. C 35S-labelling of newly synthesized mitochondrial-encoded proteins in RKO and IMT1-resistant RKO cells treated either with DMSO or with 1 μM IMT1 for four days. Left: pulse labelling, right: 24 hours chase in normal medium. Putative mitochondrial proteins are reported on the right-hand side of the gel, molecular weights on the left. Coomassie staining shows protein loading. D Densiometric quantification ±SD of ND5, COX1, ND4 band from n=4 independent experiments. Statistical significance was calculated with paired t-test: RKO + IMT1 vs resistant + IMT1, pulse: *p=0,0139, chase: *p=0,0368. E qRT-PCR relative quantification of mitochondrial transcript levels of RKO and IMT1-resistant RKO cells treated with DMSO or IMT1 for 96 hours. Data represent the mean values ± SEM of n=5 experiments. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0092, MT-RNR1 *p=0,0112, MT-ATP6 **p= 0,0022. F Northern blot analyses of mitochondrial transcripts of RKO cells treated with DMSO (RKO, "IMT1 -"), IMT1 (RKO "+ IMT1") and IMT1-resistant cells (Res, "IMT1 +"). n=3 independent experiments (Repl. 1, 2 and 3) were probed for COX2, ATP6, 16S and 12S mtRNA; the nuclear-encoded 18S RNA is shown as loading control. G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028. H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.
Induction of autophagy enhances IL‐1β secretion.Figure 1(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. (B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting. (C) As in (A), assayed for IL‐18. Data represent mean values±s.d. (n≥3); *P0.05. (D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05. (E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05. (F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05. (G) Immunoblot analysis of ASC and NLRP3 knockdowns. (H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05. (I) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP-LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β. (J, K) A line fluorescence tracing from images in (I). (L) Pearson's colocalization coefficient for IL‐1β and LC3. Pearson's coefficient was derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.
Figure 4Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (A) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR (n=3). Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (B) BALF cells were pelleted and similarly analyzed for the indicated mRNAs (n=3). Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (C) BALF supernatant was also collected and analyzed by ELISA for IL-6, IFNα and TNFα (n=3).
Figure 2(A, B) Quantitation of dendrite morphology of Trif mutant neurons (A) and Myd88-/- neurons (B) after poly(I:C) stimulation. Trif mutant or Myd88-/- neurons were transfected with a GFP construct at 4 DIV and treated with poly(I:C) one day later. Neuron morphology was analyzed at 6 DIV according to the GFP signal. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by unpaired t-test. (C) MYD88 but not TRIF expression reduces dendrite outgrowth. HA-tagged MYD88 and TRIF were co-transfected with a GFP construct into cultured wild-type (WT) neurons at 4 DIV, and cell morphology was analyzed at 6 DIV. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by one-way ANOVA with Bonferroni's multiple comparison test. (D) MYD88 knockdown, but not TRIF knockdown, robustly increases dendritic arbors and loses the response to poly(I:C). Three DIV neurons were transfected with control shRNA (shCtrl), MYD88 shRNA (shMYD88) or TRIF shRNA (shTRIF) and poly(I:C) was applied into the culture at 5 DIV for 24 h before harvest. Mean values SEM of representatives of three independent experiments are shown. The numbers of analyzed neurons in the representative experiments are indicated in each column. Data were analyzed by two-way ANOVA with Bonferroni's multiple comparison test. Scale bar, 20 μm. *** P < 0.0001.
Figure 4(A) Domain diagram of S. cerevisiae Hrr25 (top) and Mam1 (bottom), with a schematic of Mam1 secondary structure. Zinc-coordinating cysteine and histidine residues (Cys114, His116, Cys119, and Cys152) are shown in their approximate locations in the secondary structure. See Figure EV1B for Mam1 sequence alignment. (B) Overall structure of the S. cerevisiae Hrr251-394 K38R:Mam187-191 complex, colored as in (A). View is equivalent to Figure 2B. (C) Close-up view of Mam187-191, colored as in the schematic in (A). (D) Close-up of the variant zinc knuckle motif of Mam1, with zinc anomalous difference electron density shown at 3 σ (gray) and 10 σ (red). (E) Geometry of zinc binding in Mam1. Zn-S bonds and the Zn-N bond were restrained to ~2.3 Å and ~2.0 Å, respectively, during refinement.