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1.  Overcoming the false-minima problem in direct methods: structure determination of the packaging enzyme P4 from bacteriophage ϕ13 
The problems encountered during the phasing and structure determination of the packaging enzyme P4 from bacteriophage ϕ13 using the anomalous signal from selenium in a single-wavelength anomalous dispersion experiment (SAD) are described. The oligomeric state of P4 in the virus is a hexamer (with sixfold rotational symmetry) and it crystallizes in space group C2, with four hexamers in the crystallographic asymmetric unit. Current state-of-the-art ab initio phasing software yielded solutions consisting of 96 atoms arranged as sixfold symmetric clusters of Se atoms. However, although these solutions showed high correlation coefficients indicative that the substructure had been solved, the resulting phases produced uninterpretable electron-density maps. Only after further analysis were correct solutions found (also of 96 atoms), leading to the eventual identification of the positions of 120 Se atoms. Here, it is demonstrated how the difficulties in finding a correct phase solution arise from an intricate false-minima problem.
PMCID: PMC1832085  PMID: 16131757
2.  Structure of murine angiogenin 
Angiogenin is an unusual member of the pancreatic ribonuclease superfamily that induces blood vessel formation and is a promising anticancer target. The three-dimensional structure of murine angiogenin (mAng) has been determined by X-ray crystallography. Two structures are presented, one a complex with sulphate ions (1.5 Å-resolution) and the other with phosphate ions (1.6 Å-resolution). Residues forming the putative B1, P1 and B2 subsites occupy positions similar to their hAng counterparts and are likely to play similar roles. The anions occupy the P1 subsite, sulphate binding conventionally and phosphate adopting two orientations, one of which is novel. The B1 subsite is obstructed by Glu116 and Phe119, with the latter assuming a less invasive position than its hAng counterpart. Hydrophobic interactions between the C-terminal segment and the main body of the protein are more extensive than in hAng and may underly the lower enzymatic activity of the murine protein. Elsewhere, the structure of the H3–B2 loop supports the view that hAng Asn61 interacts directly with cell surface molecules and does not merely stabilize adjacent regions of the hAng structure. mAng crystals appear to offer small-molecule inhibitors a clear route to the active site and may even withstand a reorientation of the C-terminal segment that provides access to the cryptic B1 subsite. These features represent considerable advantages over crystalline hAng and bAng.
PMCID: PMC1780170  PMID: 16301790
3.  Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å. 
A multi-subunit mitochondrial membrane protein complex involved in the Krebs Cycle and respiratory chain has been crystallized in a form suitable for near-atomic resolution structure determination.
A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Å with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.
PMCID: PMC1540442  PMID: 15805592
Succinate dehydrogenase; respiration; protein structure; membrane protein; protein complex; respiratory enzyme; oxidoreductase; E.C.
4.  A robust bulk-solvent correction and anisotropic scaling procedure 
A robust method for determining bulk-solvent and anisotropic scaling parameters for macromolecular refinement is described. A maximum-likelihood target function for determination of flat bulk-solvent model parameters and overall anisotropic scale factor is also proposed.
A reliable method for the determination of bulk-solvent model parameters and an overall anisotropic scale factor is of increasing importance as structure determination becomes more automated. Current protocols require the manual inspection of refinement results in order to detect errors in the calculation of these parameters. Here, a robust method for determining bulk-solvent and anisotropic scaling parameters in macromolecular refinement is described. The implementation of a maximum-likelihood target function for determining the same parameters is also discussed. The formulas and corresponding derivatives of the likelihood function with respect to the solvent parameters and the components of anisotropic scale matrix are presented. These algorithms are implemented in the CCTBX bulk-solvent correction and scaling module.
PMCID: PMC2808320  PMID: 15983406
bulk-solvent correction; anisotropic scaling

Results 1-4 (4)