Pitfalls of molecular replacement: The structure determination of an immunoglobulin light-chain dimer
- Argonne National Lab., IL (United States)
- Univ. of Tennessee Medical Center, Knoxville, TN (United States)
The structure of protein Cle, a human light-chain dimer from the {lambda}III subgroup, was determined using 2.6 {angstrom} data; the R value is 18.4%. The structure was solved, after a false start, by molecular replacement with the {lambda}II/V Mcg protein as a search structure. When the refinement did not proceed beyond an R value of 27%, it was discovered that while the constant domains were in their correct positions in the unit cell, the incorrect variable domains were used for defining the molecule. The correct solution required a rotation of 180{degrees} around the local twofold axis that relates the two constant domains of the dimer. The correct variable domain positions overlap about 70% of the same volume as the incorrect ones of a symmetry-related molecule. The refinement distorted the geometries of the domains. Though the constant domains were in their correct positions, the r.m.s. (root-mean-square) deviation of the C{alpha} atom position was 1.2 {angstrom} when the two constant domains were compared. For the correct structure, this value is 0.5 {angstrom}. The {var_phi} and {upsilon} angles, the r.m.s. chiral value and the free R value, even when calculated a posteriori, were good indicators of the correctness of the structure. The quaternary structure of the Cle molecule is similar to that in Mcg (crystallized from ammonium sulfate); the elbow bend is 115{degrees}. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 {angstrom} closer than in Mcg or variable dimer Rei. The hydrogen bonding at the interface of the two domains is novel. Residues Tyr36 from both monomers form a hydrogen bond that is part of a network with the Gln89 residues from both monomers. For the first time hydrogen bonds were observed between the main-chain peptide N and O atoms of the complementarity-determining region CDR2 and CDR3 segments of both monomers.
- Research Organization:
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- W-31109-ENG-38
- OSTI ID:
- 625681
- Journal Information:
- Acta Crystallographica, Vol. D52; Other Information: PBD: 1996
- Country of Publication:
- Denmark
- Language:
- English
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