Hunt John F, Deisenhofer Johann
Howard Hughes Medical Institute and Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9050, USA.
Acta Crystallogr D Biol Crystallogr. 2003 Feb;59(Pt 2):214-24. doi: 10.1107/s0907444902018930. Epub 2003 Jan 23.
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.
对于SecA转位ATP酶晶体，实验相位仅能解析到4.4埃的分辨率。利用晶体65%的溶剂含量对这些相位进行密度修正，得到了一个电子密度图，据此可以构建出约80%结构的近似主链模型。将从这个部分模型推断出的相位与多波长反常散射（MIR）相位相结合，并重复密度修正，得到了一个改进的电子密度图，据此可以构建出更完整的主链模型。然而，这一过程在得到一个能对大多数蛋白质分子进行明确序列归属的电子密度图之前就收敛了。为了避免与推测性序列归属尝试相关的可能的模型偏差，采用了一种实空间交叉验证程序，以促进基于部分模型定相的晶体结构的完成。该蛋白质被划分为两个不相交的残基集。为其中一个残基集构建侧链的模型用于相位组合和密度修正，以便为未包含在模型中的另一个残基集中的残基解释产生改进的电子密度。因此，两个残基集中的残基交替从模型中省略，除非在基于与另一个残基集定相可以明确确定侧链的位点。这种乒乓交叉验证程序使得可以使用部分模型定相来完成SecA的晶体结构，而不受模型偏差的阻碍。这些结果表明，基于对部分模型定相和密度修正的大量使用，仅用低分辨率实验相位信息就可以解析大蛋白质分子的结构。实空间交叉验证可用于降低与部分模型定相相关的模型偏差风险，简化这种方法并扩大其适用范围。
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