The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

We attempted to solve the structure by molecular replacement into the P2 1 form using the NMR coordinates of a subdomain comprising SARS-CoV Nsp3 residues 513-651; PDB code 2JWJ [29, 63] ), which is almost identical to the SUD-M subdomain of SARS-CoV Nsp3. Using the program Phaser [64, 65] , we could find two solutions, and the C-terminal part of SUD core was well defined in the electron-density maps. However, for the N-terminal half, only a few segments of poly(Ala) chain could be built into the maps. This starting model was then refined in BUSTER-TNT [66] using Local Structure Similarity Restraints (LSSR) [67] as non-crystallographic symmetry (NCS) restraints to give R and R free values of 0.453 and 0.479, respectively. The resulting 2mF o -DF c electron density was subjected to density modification using solvent flattening, histogram matching, and 2-fold NCS-averaging using DM [68] . The averaging masks were calculated and updated using the auto-correlation procedure [69] as implemented in DM. Using the automatic building program BUCCANEER [70] together with REFMAC [71] (as implemented in the CCP4i [72] interface for CCP4) in an iterative procedure for 20 cycles resulted in a model for 501 residues in 10 chains (the longest having 208 residues), in which 448 residues were assigned both a chemical identity and a sequential residue number, while the remaining 53 residues were modeled as poly(Ala) in 8 shorter chains. The R and R free values resulting from REFMAC were 0.374 and 0.414, respectively. This model was refined in BUSTER-TNT, again using LSSR as NCS restraints for the common parts in the already sequenced 448 residues of the dimer, to R and R free values of 0.269 and 0.316. The improved electron density was again subjected to density modification using DM as detailed above, but using a lower solvent content of 35% as well as anisotropically scaled observed amplitudes as output by BUSTER-TNT. The resulting density-modified and NCS-averaged map was then used for automatic model building using the iterative BUCCANEER/REFMAC procedure described above. This produced a model with 511 residues in 5 chains with 487 residues sequenced. The R and R free values from REFMAC for this model were 0.289 and 0.326, respectively.