Reliable quality-control methods for protein crystal structures

Badger, John; Hendle, J.

doi:10.1107/s0907444901020133

Cited by 14 publications

(9 citation statements)

References 20 publications

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“…No sigma cutoff was included in the refinement and both structures were verified using composite omit electron density maps. The final R factors (work/free/all reflections) of 21.4/26.1/21.6 and 20.6/25.5/21.0 for K252c-soaked and CPA-soaked structures, respectively, are within the typically ranges for structures at or above 2.3 Å resolutions (Badger and Hendle, 2002). In both structures, the 20-residue N-terminal tags containing the His 6 affinity sites are disordered.…”

Section: Methodsmentioning

confidence: 72%

An Unusual Role for a Mobile Flavin in StaC-like Indolocarbazole Biosynthetic Enzymes

Goldman

Ryan

Hamill

et al. 2012

Chemistry & Biology

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SUMMARY The indolocarbazole biosynthetic enzymes StaC, InkE, RebC, and AtmC mediate the degree of oxidation of chromopyrrolic acid on route to the natural products staurosporine, K252a, rebeccamycin, and AT2433-A1, respectively. Here we show that StaC and InkE, which mediate a net 4-electron oxidation, bind FAD with a micromolar Kd, whereas RebC and AtmC, which mediate a net 8-electron oxidation, bind FAD with a nanomolar Kd, while displaying the same FAD redox properties. We further create RebC-10x, a RebC protein with ten StaC-like amino acid substitutions outside of previously characterized FAD binding motifs and the complementary StaC-10x. We find that these mutations mediate both FAD affinity and product specificity, with RebC-10x displaying higher StaC activity than StaC itself. X-ray structures of this StaC catalyst identify the substrate of StaC as 7-carboxy-K252c and suggest a unique mechanism for this FAD-dependent enzyme.

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Section: Methodsmentioning

confidence: 72%

An Unusual Role for a Mobile Flavin in StaC-like Indolocarbazole Biosynthetic Enzymes

Goldman

Ryan

Hamill

et al. 2012

Chemistry & Biology

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“…The resulting atomic model was refined using REFMAC (23) with iterative model building using XTALVIEW/XFIT. The stereochemical quality of the atomic model was monitored using an automated quality control procedure (26). The structures of Gleevec and staurosporine bound Syk were determined as above using the apo Syk-KD structure as the molecular replacement search model.…”

Section: Methodsmentioning

confidence: 99%

A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Atwell

Adams

Badger

et al. 2004

Journal of Biological Chemistry

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186

161

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Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine 3 phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.Syk family members include two human proteins, Syk (spleen tyrosine kinase) and its closest relative Zap-70 (70-kDa chain-associated protein 1). Syk operates downstream of the B-cell receptor in B-cells, the IgE receptor Fc⑀RI in mast cells, Fc␥R in macrophages (2), and other receptors (3). Zap-70 performs a similar function in T-cell receptor signaling (4). Syk is expressed in a wide range of cell types, although its function is best understood in hematopoietic cells. Knock-out mouse studies have shown that Syk is essential for lymphocyte development (2). Syk has attracted particular interest as a therapeutic target for treatment of asthma, because Syk-deficient mast cells do not degranulate in response to Fc⑀RI aggregation (5, 6). Analyses of three-dimensional structures of the kinase domains of the insulin receptor kinase (7,8), fibroblast growth factor receptor 1 (9), and Lck (10) gave rise to a model of conformational transition upon activation in kinases. In this model, the preactivated conformation is characterized by activation loop occlusion of the ATP-and/or substrate-binding sites ("loop in"), effectively preventing substrate access. Phosphorylation stabilizes an open conformation of the activation loop ("loop out") that does not occlude the substrate-binding sites and is compatible with catalysis (11, 12). In the loop-out conformation, the activation loop also forms a platform for peptide substrate docking (12). In addition to permitting access to the su...

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“…Another analysis 31 suggested that significant errors that are relatively simple to detect and fix still remain in models newly deposited at the PDB. The analysis used a set of global and local quality control criteria incorporating checks between the model and the computed electron density as well as stereochemical quality indicators.…”

Section: Significant Errors That Remainmentioning

confidence: 99%