Ping-pong cross-validation in real space: a method for increasing the phasing power of a partial model without risk of model bias.
Experimental phases could only be obtained to 4.4 A resolution for crystals of the SecA translocation ATPase. Density modification of these phases exploiting the 65% solvent content of the crystal produced a map from which an approximate backbone model could be built for 80% of the structure. Combining the phases inferred from this partial model with the MIR phases and repeating the density modification produced an improved map from which a more complete backbone model could be built. However, this procedure converged before yielding a map, that allowed unambiguous sequence assignment for the majority of the protein molecule. In order to avoid the likely model bias associated with a speculative attempt at sequence assignment, a real-space cross-validation procedure was employed to facilitate completion of the crystal structure based on partial model phasing. The protein was partitioned into two disjoint sets of residues. Models in which the side chains were built for residues in one of the two sets were used for phase combination and density modification in order to produce improved electron density for interpretation of residues in the other set that had not been included in the model. Residues in the two sets were therefore omitted from the model in alternation except at sites where the side chain could be identified definitively based on phasing with the other set. This ping-pong cross-validation procedure allowed partial model phasing to be used to complete the crystal structure of SecA without being impeded by model bias. These results show that the structure of a large protein molecule can be solved with exclusively low-resolution experimental phase information based on intensive use of partial model phasing and density modification. Real-space cross-validation can be applied to reduce the risk of model bias associated with partial model phasing, streamlining this approach and expanding its range of applicability.
Hunt
John F
JF
Howard Hughes Medical Institute and Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9050, USA. hunt@sid.bio.columbia.edu
Deisenhofer
Johann
J
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins; D018360:Crystallography, X-Ray / Q000379:methods*; D004583:Electrons; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005583:Fourier Analysis; D007091:Image Processing, Computer-Assisted / Q000379:methods; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D015203:Reproducibility of Results; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D055672:Static Electricity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins