RESUMO
Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100â K. A protein-ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.
Assuntos
Cristalografia/métodos , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimologia , Termolisina/química , Cristalização/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Geobacillus stearothermophilus/metabolismo , Ligantes , Modelos Moleculares , Conformação Proteica , Síncrotrons , Termolisina/metabolismoRESUMO
Crystals of a diatom-infecting virus (CtenRNAV) that diffracted to a resolution of 4.0â Å were grown in a mixture of 2-methyl-2,4-pentanediol (MPD), calcium chloride and sodium acetate. It was possible to freeze the crystals directly at liquid-nitrogen temperature as the reservoir solution, which included about 30% MPD, acted as a cryoprotectant during X-ray diffraction data collection. A data set was collected from a single frozen crystal obtained using this method. The crystals belonged to space group P6(3)22, with unit-cell parameters a = b = 448.67, c = 309.76â Å and two virus particles in the unit cell. The virus-particle orientation was determined using a rotation function and the virus-particle centre was estimated on the basis of crystallographic considerations. The packing of CtenRNAV in the crystal lattice was revealed by this preliminary crystallographic study.
Assuntos
Vírus de RNA/química , Proteínas Virais/química , Cristalização , Cristalografia por Raios X , Diatomáceas/virologia , Vírion/químicaRESUMO
The single segment, double-stranded RNA genome of the L-A virus (L-A) of yeast encodes two proteins: the major coat protein Gag (76 kDa) and the Gag-Pol fusion protein (180 kDa). The icosahedral L-A capsid is formed by 120 copies of Gag and has architecture similar to that seen in the reovirus, blue tongue virus and rice dwarf virus inner protein shells. Gag chemically removes the m7GMP caps from host cellular mRNAs. Previously we identified a trench on the outer surface of Gag that included His154, to which caps are covalently attached. Here we report the refined L-A coordinates at 3.4 angstroms resolution with additional structural features and the structure of L-A with bound m7GDP at 6.5 angstroms resolution, which shows the conformational change of the virus upon ligand binding. Based on site-directed mutations, residues in or adjacent to the trench that are essential (or dispensable) for the decapping reaction are described here. Along with His154, the reaction requires a cluster of positive charge adjoining the trench and residues Tyr 452, Tyr150 and either Tyr or Phe at position 538. A tentative mechanism for decapping is proposed.
Assuntos
Capsídeo/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Produtos do Gene gag/metabolismo , Capuzes de RNA/metabolismo , Vírus de RNA/química , RNA de Cadeia Dupla/metabolismo , Replicação Viral , Sítios de Ligação , Capsídeo/química , Cristalografia por Raios X , Produtos do Gene gag/química , Produtos do Gene gag/genética , Histidina/química , Histidina/genética , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Fenilalanina/química , Fenilalanina/genética , Capuzes de RNA/isolamento & purificação , Vírus de RNA/genética , Vírus de RNA/metabolismo , RNA de Cadeia Dupla/isolamento & purificação , Tirosina/química , Tirosina/genéticaRESUMO
The structure of the yeast L-A virus was determined by X-ray crystallography at 3.4 A resolution. The L-A dsRNA virus is 400 A in diameter and contains a single protein shell of 60 asymmetric dimers of the coat protein, a feature common among the inner protein shells of dsRNA viruses and probably related to their unique mode of transcription and replication. The two identical subunits in each dimer are in non-equivalent environments and show substantially different conformations in specific surface regions. The L-A virus decaps cellular mRNA to efficiently translate its own uncapped mRNA. Our structure reveals a trench at the active site of the decapping reaction and suggests a role for nearby residues in the reaction.