Crystallography &amp; NMR System: A New Software Suite for Macromolecular Structure Determination

Brünger, A.T.; Adams, Paul D.; Clore, G. Marius; DeLano, Warren L.; Gros, Piet; Grosse‐Kunstleve, Ralf W.; Jiang, Jiansheng; Kuszewski, John; Nilges, Michaël; Pannu, Navraj S.; Read, Randy J.; Rice, Luke M.; Simonson, Thomas; Warren, Gregory L.

doi:10.1107/s0907444998003254

Cited by 15,911 publications

(12,315 citation statements)

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“…We used a combination of commonly used prediction methods to determine secondary structure predictions (PSIPRED 30 and PolyPhobius 31 ) together with secondary structure propensity computed directly from local ECs 29 resulting in the identification 10 TM helices and 3 smaller helices in ECL4 and 2 beta-strands in ECL2. A total of 220 folded models for T. thermophilus RodA were generated for increasing numbers of EC restraints with using the folding protocol in EVfold 9 which itself uses a distance geometry and simulated annealing protocol in CNS 32,33 . All models were ranked as described in previously 11,34 and the 50 top-ranked models from each of the MSAs were used as as molecular replacement search models ( vide infra ).…”

Section: Methodsmentioning

confidence: 99%

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Sjodt

Brock

Dobihal

et al. 2018

Nature

120

139

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The Shape, Elongation, Division, and Sporulation (“SEDS”) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was long enigmatic, but recent work1–3 has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase – a role previously attributed exclusively to members of the penicillin binding protein family4. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis for SEDS activity, and no structural data are available for RodA or any homolog thereof. Here, we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a novel ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating it is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

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Section: Methodsmentioning

confidence: 99%

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Sjodt

Brock

Dobihal

et al. 2018

Nature

120

139

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“…According to PROCHECK-NMR analysis, the majority of the residues have ϕ and ψ angles in the favored region (74.6%), while only 1.7% of the residues (Thr-5 and Arg-102) are in the disallowed region. The 20 DYANA structures were then further refined using CNS (22). A comparison of the structural statistics for the 20 lowest-energy DYANA and CNS structures shows minimal differences between molecules calculated from each program ( Table 2).…”

Section: Solution Structure Of Frataxinmentioning

confidence: 99%

“…Author manuscript; available in PMC 2010 April 23. of simulated annealing at 25 K, 2000 slow cooling steps to 0 K, and 10 000 steps of restrained Powell minimization in Cartesian space (anneal.inp protocol) (22).…”

Section: Supplementary Materialsmentioning

confidence: 99%

“…Interatomic structural NOE restraints were determined using three-dimensional (3D) versions of the 15 N-filtered NOESY and 13 C-filtered NOESY, while aromatic through-space correlations were determined using the 2D 1 H-1 H NOESY (deuterium exchange sample) and 3D 13 C-filtered NOESY data. A complete list of NMR spectra, the collection parameters used during data collection, and references for each experiment are listed in Table 1.Structure Determination-Frataxin structures were refined using torsion angle dynamics (TAD) within DYANA and regularized within CNS using a total of 1730 NOE inter-proton, 44 hydrogen bonding, and 144 ϕ/ψ dihedral angle restraints (21,22). Backbone dihedral angle restraints were obtained on the basis of chemical shift values using TALOS (23).…”

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confidence: 99%

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Yeast Frataxin Solution Structure, Iron Binding, and Ferrochelatase Interaction^,

et al. 2004

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The mitochondrial protein frataxin is essential for cellular regulation of iron homeostasis. Although the exact function of frataxin is not yet clear, recent reports indicate the protein binds iron and can act as a mitochondrial iron chaperone to transport Fe(II) to ferrochelatase and ISU proteins within the heme and iron-sulfur cluster biosynthetic pathways, respectively. We have determined the solution structure of apo yeast frataxin to provide a structural basis of how frataxin binds and donates iron to the ferrochelatase. While the protein's α-β-sandwich structural motif is similar to that observed for human and bacterial frataxins, the yeast structure presented in this report includes the full N-terminus observed for the mature processed protein found within the mitochondrion. In addition, NMR spectroscopy was used to identify frataxin amino acids that are perturbed by the presence of iron. Conserved acidic residues in the helix 1-strand 1 protein region undergo amide chemical shift changes in the presence of Fe(II), indicating a possible iron-binding site on frataxin. NMR spectroscopy was further used to identify the intermolecular binding interface between ferrochelatase and frataxin. Ferrochelatase appears to bind to frataxin's helical plane in a manner that includes its iron-binding interface.Frataxin, a mitochondrial protein known to participate in regulating cellular iron homeostasis (1-3), has recently been suggested to play a direct role in producing both heme and Fe-S clusters (4,5). Reduced frataxin levels are the principal cause of the autosomal recessive neurodegenerative disorder Friedreich's ataxia, affecting one in 50 000 humans † This work is supported by the American Heart Association, Midwest Affiliate (Grant 0130527Z to T.L.S.) and by the National Institutes of Health (Grant DK53953 to A.D.). ‡ NMR assignments, atomic coordinates, and chemical shift data for frataxin have been deposited (PDB entry 1XAQ, BMRB entry 11688 6,7). A cellular frataxin deficiency causes mitochondrial iron overload and impairment of both heme and Fe-S cluster biosynthesis (1,2,8). Cellular heme production is accomplished by the enzyme ferrochelatase, which utilizes iron and protoporphyrin as substrates to produce heme, and recent reports indicate frataxin binds to ferrochelatase with a nanomolar binding affinity (5,9). Frataxin has also been shown to donate iron to ferrochelatase for the completion of in vitro heme synthesis (9,10). These results suggest frataxin may act as an iron chaperone to deliver the Fe(II) required to complete cellular heme biosynthesis.Frataxin has been shown to bind iron and partially protect metal against aerobic oxidation, suggesting in vivo protein could deliver the Fe(II) required by frataxin's protein binding partners to complete heme and iron-sulfur cluster biosynthesis (11)(12)(13)(14). The presence of iron can induce aggregation in human and yeast frataxins (12,13); however, this oligomerization is dependent on solution conditions (11,15). Monomeric human and bacterial ...

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“…Promising solutions with good R-factors, reasonable correlation coefficients, and few short lattice contacts were found in both space groups. Initial refinements of the models were performed with the program CNS (29), setting aside 5% randomly selected reflections for calculating the R -free (30). While the R -factor calculated for the model in the orthorhombic space group P 2 1 2 1 2 1 dropped rapidly, it failed to drop below 35% in space group P 4 2 2 1 2.…”

Section: Methodsmentioning

confidence: 99%

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

Diop-Frimpong

Prakash²,

Rajeev³

et al. 2005

Nucleic Acids Research

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Substitution of oxygen atoms by sulfur at various locations in the nucleic acid framework has led to analogs such as the DNA phosphorothioates and 4′-thio RNA. The phosphorothioates are excellent mimics of DNA, exhibit increased resistance to nuclease degradation compared with the natural counterpart, and have been widely used as first-generation antisense nucleic acid analogs for applications in vitro and in vivo. The 4′-thio RNA analog exhibits significantly enhanced RNA affinity compared with RNA, and shows potential for incorporation into siRNAs. 2-Thiouridine (s2U) and 5-methyl-2-thiouridine (m5s2U) are natural nucleotide analogs. s2U in tRNA confers greater specificity of codon–anticodon interactions by discriminating more strongly between A and G compared with U. 2-Thio modification preorganizes the ribose and 2′-deoxyribose sugars for a C3′-endo conformation, and stabilizes heteroduplexes composed of modified DNA and complementary RNA. Combination of the 2-thio and sugar 2′-O-modifications has been demonstrated to boost both thermodynamic stability and nuclease resistance. Using the 2′-O-[2-(methoxy)ethyl]-2-thiothymidine (m5s2Umoe) analog, we have investigated the consequences of the replacement of the 2-oxygen by sulfur for base-pair geometry and duplex conformation. The crystal structure of the A-form DNA duplex with sequence GCGTAT*ACGC (T* = m5s2Umoe) was determined at high resolution and compared with the structure of the corresponding duplex with T* = m5Umoe. Notable changes as a result of the incorporation of sulfur concern the base-pair parameter ‘opening’, an improvement of stacking in the vicinity of modified nucleotides as measured by base overlap, and a van der Waals interaction between sulfur atoms from adjacent m5s2Umoe residues in the minor groove. The structural data indicate only minor adjustments in the water structure as a result of the presence of sulfur. The observed small structural perturbations combined with the favorable consequences for pairing stability and nuclease resistance (when combined with 2′-O-modification) render 2-thiouracil-modified RNA a promising candidate for applications in RNAi.

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Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination

Cited by 15,911 publications

References 50 publications

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Yeast Frataxin Solution Structure, Iron Binding, and Ferrochelatase Interaction^,

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

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Crystallography & NMR System: A New Software Suite for Macromolecular Structure Determination

Cited by 15,911 publications

References 50 publications

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Yeast Frataxin Solution Structure, Iron Binding, and Ferrochelatase Interaction,

Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2'-O-[2-(methoxy)ethyl]-2-thiothymidines

Contact Info

Product

Resources

About

Yeast Frataxin Solution Structure, Iron Binding, and Ferrochelatase Interaction^,