Pitfalls of molecular replacement: The structure determination of an immunoglobulin light-chain dimer

Journal Article · Tue Dec 31 04:00:00 EST 1996 · Acta Crystallographica

DOI:https://doi.org/10.1107/S090744499600813X· OSTI ID:625681

Huang, D B; Ainsworth, C; Schiffer, M ^[1]; Solomon, A ^[2]

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1. Argonne National Lab., IL (United States)
2. 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 Laboratory (ANL), Argonne, IL
• Sponsoring Organization: USDOE
• DOE Contract Number: W-31109-ENG-38
• OSTI ID: 625681
• Journal Information: Acta Crystallographica, Journal Name: Acta Crystallographica Vol. D52; ISSN ACCRA9; ISSN 0365-110X
• Country of Publication: Denmark
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
CHEMICAL BONDS
DIMERS
MAN
MONOMERS
PROTEIN STRUCTURE