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Improved crystallographic structures using extensive combinatorial refinement.

ABSTRACT: Identifying errors and alternate conformers and modeling multiple main-chain conformers in poorly ordered regions are overarching problems in crystallographic structure determination that have limited automation efforts and structure quality. Here, we show that implementation of a full factorial designed set of standard refinement approaches, termed ExCoR (Extensive Combinatorial Refinement), significantly improves structural models compared to the traditional linear tree approach, in which individual algorithms are tested linearly and are only incorporated if the model improves. ExCoR markedly improved maps and models and reveals building errors and alternate conformations that were masked by traditional refinement approaches. Surprisingly, an individual algorithm that renders a model worse in isolation could still be necessary to produce the best overall model, suggesting that model distortion allows escape from local minima of optimization target function, here shown to be a hallmark limitation of the traditional approach. ExCoR thus provides a simple approach to improving structure determination.

SUBMITTER: Nwachukwu JC

PROVIDER: S-EPMC4070946 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Improved crystallographic structures using extensive combinatorial refinement.

Nwachukwu Jerome C JC Southern Mark R MR Kiefer James R JR Afonine Pavel V PV Adams Paul D PD Terwilliger Thomas C TC Nettles Kendall W KW

Structure (London, England : 1993) 20130926 11

Identifying errors and alternate conformers and modeling multiple main-chain conformers in poorly ordered regions are overarching problems in crystallographic structure determination that have limited automation efforts and structure quality. Here, we show that implementation of a full factorial designed set of standard refinement approaches, termed ExCoR (Extensive Combinatorial Refinement), significantly improves structural models compared to the traditional linear tree approach, in which indi ...[more]

PMID: 24076406

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Project description:A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

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Improved crystallographic structures using extensive combinatorial refinement.

Publications

Improved crystallographic structures using extensive combinatorial refinement.

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