Pitfalls of molecular replacement

The structure determination of an immunoglobulin light-chain dimer

D. B. Huang, C. Ainsworth, Alan Solomon, M. Schiffer

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The structure of protein Cle, a human light-chain dimer from the λIII subgroup, was determined using 2.6 Å data; the R value is 18.4%. The structure was solved, after a false start, by molecular replacement with the λII/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 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 Cα atom position was 1.2 Å when the two constant domains were compared. For the correct structure, this value is 0.5 Å. The φ and ψ 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°. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 Å 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.

Original languageEnglish (US)
Pages (from-to)1058-1066
Number of pages9
JournalActa Crystallographica Section D: Biological Crystallography
Volume52
Issue number6
DOIs
StatePublished - Dec 1 1996
Externally publishedYes

Fingerprint

Immunoglobulin Light Chains
Dimers
Hydrogen
dimers
Complementarity Determining Regions
Hydrogen bonds
Monomers
Ammonium Sulfate
Hydrogen Bonding
Elbow
Molecules
Proteins
Light
Atoms
Peptides
monomers
Geometry
hydrogen bonds
proteins
molecules

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics
  • Clinical Biochemistry
  • Structural Biology
  • Condensed Matter Physics

Cite this

Pitfalls of molecular replacement : The structure determination of an immunoglobulin light-chain dimer. / Huang, D. B.; Ainsworth, C.; Solomon, Alan; Schiffer, M.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 52, No. 6, 01.12.1996, p. 1058-1066.

Research output: Contribution to journalArticle

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abstract = "The structure of protein Cle, a human light-chain dimer from the λIII subgroup, was determined using 2.6 {\AA} data; the R value is 18.4{\%}. The structure was solved, after a false start, by molecular replacement with the λII/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 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 Cα atom position was 1.2 {\AA} when the two constant domains were compared. For the correct structure, this value is 0.5 {\AA}. The φ and ψ 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°. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 {\AA} 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.",
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AB - The structure of protein Cle, a human light-chain dimer from the λIII subgroup, was determined using 2.6 Å data; the R value is 18.4%. The structure was solved, after a false start, by molecular replacement with the λII/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 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 Cα atom position was 1.2 Å when the two constant domains were compared. For the correct structure, this value is 0.5 Å. The φ and ψ 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°. However, the arrangement of the variable domains differs from that observed in other variable domain dimers. The variable domains of Cle are 0.7 Å 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.

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