human ea3a 8 human full length a3b  (Addgene inc)

Journal: Science Advances

Article Title: Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition

doi: 10.1126/sciadv.abm2827

Figure Lengend Snippet: ( A ) Overlay of the structures of BORF2 (blue), human RNR α subunit (PDB: 6aui, chain A; tan), and E. coli RNR α subunit (PDB: 6w4x, chain B; green). Novel BORF2 insertions SHI and LLI are depicted in red. Loop regions surrounding the A3Bctd active site are labeled L1, L3, and L7. ( B ) Schematic of BORF2 (1 to 826 amino acids) showing the A3B interacting regions (orange) and the novel SHI and LLI insertions (red). Bottom left: Structural comparison of the BORF2 SHI and the corresponding regions of human and E. coli RNR α subunits. Bottom right: Cryo-EM map of the BORF2 SHI domain in blue. ( C ) Close-up of BORF2-A3B interface showing a network of interactions. BORF2 is depicted in blue, BORF2 SHI is in red, and A3Bctd is in orange, with ribbon and surface representations (top and bottom, respectively). ( D ) Co-IP reactions with the indicated BORF2 constructs (anti-FLAG) and A3Bctd-eGFP. A parallel reaction with BORF2 and A3Gctd-eGFP is shown as an additional negative control. Input blots are shown below including anti-tubulin as a loading control.

Article Snippet: Full-length A3B-eGFP, A3Bctd 193–382 -eGFP, full-length A3A-eGFP, and A3Gctd 197–384 -eGFP used in localization experiments ( and and figs. S7 and S9) and co-IP experiments ( and and fig. S6) were cloned into pCDNA5TO (Invitrogen) using PCR amplification, restriction enzyme digestion, and ligation.

Techniques: Labeling, Cryo-EM Sample Prep, Co-Immunoprecipitation Assay, Construct, Negative Control