Computational protocol: Crystal structure of the Rous sarcoma virus intasome

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Protocol publication

[…] X-ray diffraction data were collected at the Advanced Photon Source Northeastern Collaborative Access Team beamlines (24-ID-C/E) and the Advanced Light Source Molecular Biology Consortium (4.2.2) beamline and processed using HKL2000 or XDS . The RSV intasome crystals showed varied degrees of pseudo-merohedral twinning with twin operator [l, −k, h], owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice. Thus, we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions. The structure of RSV intasome was determined by molecular replacement with PHASER , using the RSV IN CCD, CTD (PDB ID: 4FW1) , and a 16 bp B-form DNA as search models. 8 copies of CCD, one copy of CTD and three copies of DNA molecules were located. Refinement of the partial model revealed electron density for two copies of the metatungstate clusters. The metatungstate clusters were placed into the electron density by molecular replacement using MOLREP . Subsequent iterative model building using COOT and refinement with PHENIX suite allowed placement of the remaining 7 copies of CTD, 8 copies of NTD generated using MODELLER based on HIV-1 NTD (PDB ID: 1K6Y) , and building of the inter-domain linkers as well as the remaining parts of the DNA molecule guided by the difference electron density maps. A third metatungstate cluster, which is more weakly bound compared to the first two, was positioned manually into residual density. The DNA base-pairs and base-stacking restraints were used throughout the refinement. The geometry restraints for protein included the reference-model restraints for CCD and CTD based on the higher resolution RSV IN structure (4FW1) , and the secondary structure and zinc-coordination restraints for NTD. Atomic displacement parameters were refined with grouped B-factors per residue for protein and DNA, and a total of 53 TLS groups assigned by PHENIX . Twelve tungsten atoms representing each cluster were refined as a rigid body. The asymmetric unit of the crystal contains one complete RSV intasome, which includes eight IN molecules and two viral DNA branches emanating from a strongly bent 38 bp target DNA. The dataset used for the final refinement was from an RSV intasome crystal grown using selenomethionine-labeled RSV IN (1–270) with the following amino acid substitutions; C23S, L112M, L135M, L162M, L163M, L188M, and L189M, which we confirmed to be active in concerted integration and inhibited by INSTI similarly to the wild-type RSV IN, and a DNA substrate carrying a nick at the middle of each target DNA branch (the 16-base DNA strand shown in olive in has a nick 8 bases from either end). The nick occasionally facilitated crystal growth, but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick. Because this nick in the target DNA is not biologically relevant, it is not shown in , , to avoid confusion. Twin refinement protocol was not used as the dataset used for the final refinement had a low (less than 10%) twin fraction. The summary of data collection and refinement statistics is shown in . The paired-refinement procedure was performed in steps of 0.1 Å to determine the high-resolution limit (). The register of amino acids in the final model was verified by the selenium anomalous difference Fourier peaks (). Ramachandran analysis shows that 96.0, 3.9, and 0.1 % of the protein residues are in the most favored, allowed, and disallowed region, respectively. The NTD-CCD linker in some of the non-catalytic IN molecules and the last four bp at the distal end of one of the viral DNA molecules were not built due to poor electron density. The molecular graphics images were produced using PYMOL (www.pymol.org). […]

Pipeline specifications

Software tools XDS, Molrep, Coot, PHENIX, MODELLER, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Human immunodeficiency virus 1, Rous sarcoma virus
Diseases Sarcoma, Avian