carf  (Proteintech)

Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF is highly expressed in spermatocytes and spermatids of the testis (A) NCBI database analysis of the expression profile of Carf mRNA in mouse tissues. (B) qPCR analyses of Carf mRNA levels in multiple organs of mice. Data are presented as the mean ± SEM, n = 3. (C) Immunofluorescence staining analysis of CARF (green) in testis sections. γH2AX (red) was used as a marker for spermatocytes. The nuclei were stained with DAPI (blue). Spc indicates spermatocytes, Ser indicates Sertoli cells, Ley indicates Leydig cells, and Rspd indicates round sperm. Scale bar: 50 μm. (D) Immunofluorescence staining analysis of CARF (red) and PNA (green) in testis sections. The nuclei were stained with DAPI (blue), Scale bar: 50 μm. (E,F) The expression pattern of CARF in the mouse germline atlas was analyzed via a single-cell sequencing database (http://malehealthatlas.cn/ ).

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Expressing, Immunofluorescence, Staining, Marker, Sequencing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF interacts with PABPC1 to participate in RNA alternative splicing (A) Abnormal alternative splicing patterns, including skipped exons, alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE) and retained intron (RI) caused by Carf defects. (B) Abnormal variable splicing of the functional gene Hira, which is related to germ cell development caused by Carf defects. It belongs to the alternative 5′ splice site (A5SS) exception mode. (C) Abnormal variable splicing of the functional gene Surf1, which is related to germ cell development caused by Carf defects. It is an alternative 3′ splice site (A3SS). (D) Abnormal variable splicing of the functional gene Usf2, which is related to germ cell development caused by Carf defects. It belongs to the mutually exclusive exons (MXE). (E) Abnormal variable splicing of the functional gene Lrmp, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (F) Abnormal variable splicing of the functional gene Pkd2l1, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (G) Co-IP analysis of the interaction between CARF and PABPC1. PABPC1 expression was detected in the IP products of CARF, and IgG was used as a control. GAPDH served as a loading control. (H) Co-IP analysis of the interaction between CARF and PABPC1. CARF expression was detected in the IP products of PABPC1, and IgG was used as a control. GAPDH served as a loading control. (I) Immunostaining of PABPC1 in wild-type and Carf–/– testis sections (PABPC1: green); DAPI was used to stain the dye the nuclei, scale bar: 50 μm. (J) Western blot analysis of PABPC1 protein levels in testes from wild-type and Carf–/– mice. GAPDH served as a loading control. (K) Immunohistochemical analysis of the expression of PABPC1 in testes from wild-type and Carf–/– testis sections. Scale bar: 50 μm. (L) Quantitative results of (K). n = 3, Data are presented as the mean ± SEM. ***P < 0.001.

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Alternative Splicing, Functional Assay, Co-Immunoprecipitation Assay, Expressing, Control, Immunostaining, Staining, Western Blot, Immunohistochemical staining

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Domain architecture of Bacteroidales Cad1. The protein contains a N-terminal CARF domain followed by a linker (L1) and a C-terminal adenosine deaminase (ADA) domain. Residue numbers are labeled. (B) Cad1 used in this study was identified within a type III CRISPR- cas locus in a contig from an unknown Bacteroidales bacterium. (C) Association of Cad1 homologs with different subtypes of type III CRISPR-Cas systems (88 non-redundant homologs used for quantification). (D) Growth of staphylococci carrying pTarget and different pCRISPR variants, measured as OD 600 after the addition of aTc. Mean of three biological triplicates, ±SEM, is reported. (E) Enumeration of colony-forming units (CFUs) from staphylococcal cultures carrying different pCRISPR variants after the addition of aTc. At the indicated times after induction, aliquots were removed and plated on solid medium with or without aTc to count the remaining viable cells. Mean of three biological replicates, ±SEM, is reported. (F) Time course microscopy of S. aureus cells harboring pTarget and pCRISPR(Δspc) or pCRISPR(Cad1) at different times after addition of aTc, experiment repeated for two biological replicates. Scale bar, 4 μM. (G) Quantification of ITP/ATP ratios from bacterial lysates. Extracts from staphylococci harboring pTarget and pCRISPR(Δspc) or pCRISPR(Cad1) were either collected before (0 min) or after (15 min) incubation with aTc and analyzed via LC-MS. Mean of five biological replicates ±SEM, is reported. p values, obtained with a two-sided t test with Welch’s correction, are shown. See also and .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Residue, Labeling, CRISPR, Microscopy, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of cOAs associated with purified Cad1-His 6 . cA 4 and cA 6 standards were used as controls. (B) ITC binding curve of Cad1-CARF-His 6 to cA 4 and cA 6 representing the FitX and FitY values estimated by MicroCal PEAQ-ITC analysis software (Malvern). K d values are ~700 and ~30 nM, respectively. (C) Crystal structure of dimeric apo-Cad1-CARF-His 6 . The predicted ligand binding pocket is shown, and the C-terminal legs from each monomer are colored in cyan. The angle between the C-terminal legs is 58°. (D) cA 6 -bound structure of Cad1-CARF-His 6 showing the ligand at the dimeric interface of the CARF domains, with the angle between the C-terminal legs becoming 55°. (E) cA 4 -bound structure of Cad1-CARF-His 6 showing an increase in the spread of the C-terminal legs to 76°. (F) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring alanine substitutions of Cad1 residues involved in cOA binding, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Purification, Binding Assay, Software, Ligand Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Cryo-EM structure of hexameric apo-Cad1 illustrating the arrangement of AB, CD, and EF Cad1 dimers (in green, blue, and pink, respectively) aligned in a 3-fold symmetric arrangement (black center triangle). A pocket is formed on the interface of the CARF and ADA domains, and the distance between these domains is 15 Å (closed) on one side of the Cad1 dimer and increases to 24 Å (open) on the opposite side. The pocket is generated by the tilting of the CARF head domain (black dotted arrows). (B) Cryo-EM structure of ATP-bound hexameric Cad1 (conformation 1) displaying AB, CD, and EF dimers in green, blue, and pink, respectively. Insets highlight the residues at one of the inter-domain ATP binding sites (red border, with red arrowheads pointing at the ATP) and one of the empty deaminase pockets (black border). The metal is modeled as Mg +2 . (C) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring the amino acid substitutions of the residues lining the inter-domain ATP binding pocket, measured as the OD 600 value after 220 min of addition of aTc. Mean of three biological triplicates, ±SEM, is reported. (D) Cryo-EM structure of hexameric Cad1 protein in presence of ATP and cA 4 , displaying AB, CD, and EF dimers in green, blue, and pink, respectively. cA 4 , ATP, and ATP/ITP molecules are shown in space-filling representation. Insets highlight the residues at one of the inter-domain ATP binding sites (red border, with red arrowheads pointing at the ATP) and one of the deaminase pockets (black border). The metal is modeled as Mg +2 . See also , , , and .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Cryo-EM Sample Prep, Generated, Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Amino acid residues in the deaminase pocket of the apo-Cad1-ATP structure. Dotted lines indicate the distances (in Å) between side chains and the metal ion (modeled as Mg +2 ). (B) Same as (A) but for the deaminase pocket of cA 4 -Cad1-ATP. (C) Superimposition of the structures shown in (A) and (B). The shift in the H448 residue in the ATP-bound cA 4 -Cad1 structure is pointed out by a gray arrow. (D) Superposition of the deaminase pocket of the apo-Cad1-ATP structure (silver) and the cA 4 -Cad1-ATP structure (green). The shift in the loop containing H448 and E451 residues is shown by a red double arrow. The distance between the Cα atoms of E451 in the apo-Cad1-ATP structure (black arrowhead) and in the cA 4 -Cad1-ATP structure (red arrowhead) is marked by black dashed line. (E) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring the amino acid substitutions of the residues involved in ATP/ITP interactions at the deaminase site, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. (F) Cryo-EM structure of hexameric cA 6 -bound Cad1 in the presence of ATP. cA 6 was partly modeled within the CARF binding pocket due to the lack of density. ATP was modeled in three inter-domain binding sites (red border, with red arrowheads pointing at the ATP), and adenine was modeled at the other three sites (gray arrowheads). ATP/ITP and Mg +2 were modeled in four of the six deaminase pockets (black border inset) and only phosphate groups of ATP/ITP and Mg +2 in the other two due to lack of density. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Residue, Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of Cad1 (wild-type and mutant versions; 2 μM) reaction products in the presence or absence of ATP (1 mM) and different cOAs (20 μM). Chromatograms of ATP and ITP are shown as standards. Reactions were performed in duplicate. (B) Same as (A) but in the presence of ATP and 100 nM of the indicated cOA. (C) Quantification of the product peaks obtained in (B) as percent of ATP substrate deaminated by Cad1. Reactions performed in triplicate, ±SEM, are reported. p values, obtained with a two-sided t test with Welch’s correction, are shown. (D) HPLC analysis of Cad1 (2 μM) reaction products in the presence adenosine, AMP or ATP (1 mM), and cA 6 (20 μM). Chromatograms of adenosine and AMP are shown as standards. Reactions were performed in duplicate. (E) Same as (D) but using dATP as a substrate. (F) Same as (D) but using CTP as substrate. (G) HPLC chromatograms of Cad1 incubated with the indicated cOA at 500 μM. AMP is provided as a standard. Products of ring nuclease activity would be expected to run between cA 6 and AMP. (H) Quantification of the percent of ATP substrate deaminated by Cad1 incubated with different divalent cations at a concentration of 1 mM except for Zn 2+ , which was incubated at a concentration of 100 μM to mitigate oxidation-induced aggregation of Cad1. A reaction using Mg +2 was performed in the presence of 3 mM EDTA. Reactions were performed in triplicate, and areas under the curve for ATP and ITP peaks were used to determine the % deamination. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Mutagenesis, Incubation, Activity Assay, Concentration Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of reactions products of incubation of different Cad1 fractions (H, hexameric; M, megadalton) in the presence of cA 6 and ATP. Reactions were performed in duplicate. (B) Side view of the AB and CD dimers in the apo-Cad1 hexamer structure illustrating the residues present at the dimer-dimer interface. The residues mutated in this study are shown in the inset. (C) SECMALS analysis of hexameric Cad1 K342A (412 kDa ± 0.485%). (D) SECMALS analysis of hexameric Cad1 E408A (394 kDa ± 0.378%). (E) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring alanine substitutions of residues thought to be involved in the association of Cad1 dimers, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. (F) SECMALS analysis of purified dimeric Cad1 W349A (139 kDa ± 0.518%).

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Incubation, Purification

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Virus, Recombinant, Sequencing, Software

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Domain architecture of Bacteroidales Cad1. The protein contains a N-terminal CARF domain followed by a linker (L1) and a C-terminal adenosine deaminase (ADA) domain. Residue numbers are labeled. (B) Cad1 used in this study was identified within a type III CRISPR- cas locus in a contig from an unknown Bacteroidales bacterium. (C) Association of Cad1 homologs with different subtypes of type III CRISPR-Cas systems (88 non-redundant homologs used for quantification). (D) Growth of staphylococci carrying pTarget and different pCRISPR variants, measured as OD 600 after the addition of aTc. Mean of three biological triplicates, ±SEM, is reported. (E) Enumeration of colony-forming units (CFUs) from staphylococcal cultures carrying different pCRISPR variants after the addition of aTc. At the indicated times after induction, aliquots were removed and plated on solid medium with or without aTc to count the remaining viable cells. Mean of three biological replicates, ±SEM, is reported. (F) Time course microscopy of S. aureus cells harboring pTarget and pCRISPR(Δspc) or pCRISPR(Cad1) at different times after addition of aTc, experiment repeated for two biological replicates. Scale bar, 4 μM. (G) Quantification of ITP/ATP ratios from bacterial lysates. Extracts from staphylococci harboring pTarget and pCRISPR(Δspc) or pCRISPR(Cad1) were either collected before (0 min) or after (15 min) incubation with aTc and analyzed via LC-MS. Mean of five biological replicates ±SEM, is reported. p values, obtained with a two-sided t test with Welch’s correction, are shown. See also and .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Residue, Labeling, CRISPR, Microscopy, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of cOAs associated with purified Cad1-His 6 . cA 4 and cA 6 standards were used as controls. (B) ITC binding curve of Cad1-CARF-His 6 to cA 4 and cA 6 representing the FitX and FitY values estimated by MicroCal PEAQ-ITC analysis software (Malvern). K d values are ~700 and ~30 nM, respectively. (C) Crystal structure of dimeric apo-Cad1-CARF-His 6 . The predicted ligand binding pocket is shown, and the C-terminal legs from each monomer are colored in cyan. The angle between the C-terminal legs is 58°. (D) cA 6 -bound structure of Cad1-CARF-His 6 showing the ligand at the dimeric interface of the CARF domains, with the angle between the C-terminal legs becoming 55°. (E) cA 4 -bound structure of Cad1-CARF-His 6 showing an increase in the spread of the C-terminal legs to 76°. (F) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring alanine substitutions of Cad1 residues involved in cOA binding, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Purification, Binding Assay, Software, Ligand Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Cryo-EM structure of hexameric apo-Cad1 illustrating the arrangement of AB, CD, and EF Cad1 dimers (in green, blue, and pink, respectively) aligned in a 3-fold symmetric arrangement (black center triangle). A pocket is formed on the interface of the CARF and ADA domains, and the distance between these domains is 15 Å (closed) on one side of the Cad1 dimer and increases to 24 Å (open) on the opposite side. The pocket is generated by the tilting of the CARF head domain (black dotted arrows). (B) Cryo-EM structure of ATP-bound hexameric Cad1 (conformation 1) displaying AB, CD, and EF dimers in green, blue, and pink, respectively. Insets highlight the residues at one of the inter-domain ATP binding sites (red border, with red arrowheads pointing at the ATP) and one of the empty deaminase pockets (black border). The metal is modeled as Mg +2 . (C) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring the amino acid substitutions of the residues lining the inter-domain ATP binding pocket, measured as the OD 600 value after 220 min of addition of aTc. Mean of three biological triplicates, ±SEM, is reported. (D) Cryo-EM structure of hexameric Cad1 protein in presence of ATP and cA 4 , displaying AB, CD, and EF dimers in green, blue, and pink, respectively. cA 4 , ATP, and ATP/ITP molecules are shown in space-filling representation. Insets highlight the residues at one of the inter-domain ATP binding sites (red border, with red arrowheads pointing at the ATP) and one of the deaminase pockets (black border). The metal is modeled as Mg +2 . See also , , , and .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Cryo-EM Sample Prep, Generated, Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) Amino acid residues in the deaminase pocket of the apo-Cad1-ATP structure. Dotted lines indicate the distances (in Å) between side chains and the metal ion (modeled as Mg +2 ). (B) Same as (A) but for the deaminase pocket of cA 4 -Cad1-ATP. (C) Superimposition of the structures shown in (A) and (B). The shift in the H448 residue in the ATP-bound cA 4 -Cad1 structure is pointed out by a gray arrow. (D) Superposition of the deaminase pocket of the apo-Cad1-ATP structure (silver) and the cA 4 -Cad1-ATP structure (green). The shift in the loop containing H448 and E451 residues is shown by a red double arrow. The distance between the Cα atoms of E451 in the apo-Cad1-ATP structure (black arrowhead) and in the cA 4 -Cad1-ATP structure (red arrowhead) is marked by black dashed line. (E) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring the amino acid substitutions of the residues involved in ATP/ITP interactions at the deaminase site, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. (F) Cryo-EM structure of hexameric cA 6 -bound Cad1 in the presence of ATP. cA 6 was partly modeled within the CARF binding pocket due to the lack of density. ATP was modeled in three inter-domain binding sites (red border, with red arrowheads pointing at the ATP), and adenine was modeled at the other three sites (gray arrowheads). ATP/ITP and Mg +2 were modeled in four of the six deaminase pockets (black border inset) and only phosphate groups of ATP/ITP and Mg +2 in the other two due to lack of density. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Residue, Cryo-EM Sample Prep, Binding Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of Cad1 (wild-type and mutant versions; 2 μM) reaction products in the presence or absence of ATP (1 mM) and different cOAs (20 μM). Chromatograms of ATP and ITP are shown as standards. Reactions were performed in duplicate. (B) Same as (A) but in the presence of ATP and 100 nM of the indicated cOA. (C) Quantification of the product peaks obtained in (B) as percent of ATP substrate deaminated by Cad1. Reactions performed in triplicate, ±SEM, are reported. p values, obtained with a two-sided t test with Welch’s correction, are shown. (D) HPLC analysis of Cad1 (2 μM) reaction products in the presence adenosine, AMP or ATP (1 mM), and cA 6 (20 μM). Chromatograms of adenosine and AMP are shown as standards. Reactions were performed in duplicate. (E) Same as (D) but using dATP as a substrate. (F) Same as (D) but using CTP as substrate. (G) HPLC chromatograms of Cad1 incubated with the indicated cOA at 500 μM. AMP is provided as a standard. Products of ring nuclease activity would be expected to run between cA 6 and AMP. (H) Quantification of the percent of ATP substrate deaminated by Cad1 incubated with different divalent cations at a concentration of 1 mM except for Zn 2+ , which was incubated at a concentration of 100 μM to mitigate oxidation-induced aggregation of Cad1. A reaction using Mg +2 was performed in the presence of 3 mM EDTA. Reactions were performed in triplicate, and areas under the curve for ATP and ITP peaks were used to determine the % deamination. See also .

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Mutagenesis, Incubation, Activity Assay, Concentration Assay

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: (A) HPLC analysis of reactions products of incubation of different Cad1 fractions (H, hexameric; M, megadalton) in the presence of cA 6 and ATP. Reactions were performed in duplicate. (B) Side view of the AB and CD dimers in the apo-Cad1 hexamer structure illustrating the residues present at the dimer-dimer interface. The residues mutated in this study are shown in the inset. (C) SECMALS analysis of hexameric Cad1 K342A (412 kDa ± 0.485%). (D) SECMALS analysis of hexameric Cad1 E408A (394 kDa ± 0.378%). (E) Growth of staphylococci carrying pTarget and pCRISPR(Cad1) harboring alanine substitutions of residues thought to be involved in the association of Cad1 dimers, measured as the OD 600 value after 220 min of addition of aTc. Mean of four biological replicates, ±SEM, is reported. (F) SECMALS analysis of purified dimeric Cad1 W349A (139 kDa ± 0.518%).

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Incubation, Purification

Journal: Cell

Article Title: The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity

doi: 10.1016/j.cell.2024.10.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The x-ray diffraction data of the crystals of apo-Cad1-CARF, cA 6 -Cad1-CARF complex and cA 4 -Cad1-CARF complex were collected at the synchrotron beamline at Brookhaven National Lab (BNL).

Techniques: Virus, Recombinant, Sequencing, Software