| - | Nanobodies or VHHs (Variable Heavy domains of Heavy chain) are single domain antibodies that comprise three antigenic complementary determining regions (CDR). Nanobodies are used in numerous scientific applications including, bio-imaging, diagnosis, therapeutics, and macromolecular crystallography. We obtained crystals of a approximately 14 kDa nanobody specific for the NEIL1 DNA glycosylase (hereafter called A5) in 0.5 M ammonium sulfate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, and 1.0 M lithium sulfate monohydrate from the Crystal HT Hampton Research screen that were further optimized. Here, we describe the structure determination and refinement of the A5 crystals to a resolution of 2.1 A. The data collected were complicated by the presence of anisotropy and twinning, and while initial space group determination pointed to a higher apparent tetragonal crystal system, the data statistics suggested twinning, placing the crystal in an orthorhombic system. Twinning was confirmed by the Padilla and Yeates test, H-test, and Britton test based on local intensity differences with a twin fraction of 0.4. Molecular replacement produced the best solution in the orthorhombic space group P2 (1) 2 (1) 2 with four molecules in the asymmetric unit and we were able to model over 96% of the residues in the electron density with a final R (work) and R (free) of 0.1988 and 0.2289 upon refinement. SYNOPSIS: The crystal structure of a specific nanobody against NEIL1 was determined to 2.1 A. The structure was ultimately solved in an orthorhombic space group after diffraction data analysis revealed mild anisotropy as well as pseudo-merohedral twinning. | + | Nanobodies (VHHs) are single-domain antibodies with three antigenic CDR regions and are used in diverse scientific applications. Here, an approximately 14 kDa nanobody (A5) specific for the endonuclease VIII (Nei)-like 1 or NEIL1 DNA glycosylase involved in the first step of the base-excision repair pathway was crystallized and its structure was determined to 2.1 A resolution. The crystals posed challenges due to potential twinning and anisotropic diffraction. Despite inconclusive twinning indicators, reprocessing in an orthorhombic setting and molecular replacement in space group P2(1)2(1)2 enabled the successful modeling of 96% of residues in the asymmetric unit, with final R(work) and R(free) values of 0.199 and 0.229, respectively. |
| - | Deciphering the orthorhombic crystal structure of a novel NEIL1 nanobody with pseudo-merohedral twinning.,Thompson MK, Sharma N, Prakash A bioRxiv. 2023 Aug 7:2023.08.07.552313. doi: 10.1101/2023.08.07.552313. Preprint. PMID:37609231<ref>PMID:37609231</ref> | + | Deciphering the crystal structure of a novel nanobody against the NEIL1 DNA glycosylase.,Thompson MK, Sharma N, Thorn A, Prakash A Acta Crystallogr D Struct Biol. 2024 Feb 1;80(Pt 2):137-146. doi: , 10.1107/S205979832400038X. Epub 2024 Jan 30. PMID:38289715<ref>PMID:38289715</ref> |
| | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
