In-house phase determination of the lima bean trypsin inhibitor: a low-resolution sulfur-SAD case

Debreczeni, J.; Bunkóczi, Gábor; Girmann, Beatrix; Sheldrick, George M.

doi:10.1107/s0907444902020917

Cited by 49 publications

(53 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since the crystals exhibited hemihedral twinning (19) with a twinning fraction of around 0.2, the diffraction intensities were detwinned using DETWIN. Initial selenium sites were located from data collected at peak and remote wavelengths using SHELXD and SHELXE (20) and refined with SHARP (21). Solvent flattening, histogram matching, and 2-fold noncrystallographic symmetric were applied in DM (22) to improve the phases.…”

Section: Protein Purification and Crystallization-fmdv 3cmentioning

confidence: 99%

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

Birtley¹,

Knox

Jaulent

et al. 2005

Journal of Biological Chemistry

133

144

View full text Add to dashboard Cite

Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C pro ), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C pro , determined the crystal structure to 1.9-Å resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C pro adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4 -P4) and that FMDV 3C pro discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C pro enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C pro of an extended ␤-ribbon that folds over the substrate binding cleft in other picornavirus 3C pro structures. Collectively, these results establish a valuable framework for the development of FMDV 3C pro inhibitors.

show abstract

Section: Protein Purification and Crystallization-fmdv 3cmentioning

confidence: 99%

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

Birtley¹,

Knox

Jaulent

et al. 2005

Journal of Biological Chemistry

133

144

View full text Add to dashboard Cite

show abstract

“…The anomalous scattering of sulfur atoms was used in the classic structure determination of crambin (29). Since then, sulfur anomalous scattering has contributed to a number of structure determinations (30), although only a few of the new structures have resulted from sulfur anomalous scattering data collected on a rotating anode source rather than at a synchrotron (31)(32)(33)(34). The primary limitation of this approach is the small anomalous signal obtained from sulfur with conventional copper anode targets.…”

mentioning

confidence: 99%

Crystal Structure of the Oxygen-dependant Coproporphyrinogen Oxidase (Hem13p) of Saccharomyces cerevisiae

Phillips¹,

Whitby

Warby³

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded antiparallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a ␤-ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the ϳ8-Å movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.

show abstract

“…SAD phasing has been reported at remote wavelengths above the absorption edge (Leonard et al, 2005) and even at Cu Kα edge of in-house X-ray sources (Dauter et al, 2002;Debreczeni et al, 2003a;2003c;Evans and Bricogne, 2003;Roeser et al, 2005;Sarma and Karplus, 2006;Stevenson et al, 2004;Vennila and Velmurugan, 2011;Yogavel et al, 2007;2010a;2010b). The successful in-house SAD phasing opens new avenue of experimental phasing with in-house X-ray sources.…”

Section: Introductionmentioning

confidence: 98%