[33] AMoRe: An automated molecular replacement program package

Navaza, Jorge; Saludjian, P.

doi:10.1016/s0076-6879(97)76079-8

Cited by 552 publications

(434 citation statements)

References 17 publications

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“…The substrate-free homology model yielded putative solutions for the rotation and translation functions of the ADP form, first using the CNS (crystallography and NMR system) program (45) and then confirmed with Phaser (46,47) and AMoRe, both from the CCP4 suite (48). Following a preliminary refinement of the ADP complex structure, it was then possible to find a molecular replacement solution for the apoform structure that was consistent with the 2-fold noncrystallographic symmetry revealed in its self-rotation function.…”

Section: Methodsmentioning

confidence: 95%

The Structure of Lombricine Kinase

Bush

Kirillova

Clark

et al. 2011

Journal of Biological Chemistry

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Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309 -317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His 178 . Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.Lombricine, arginine, and creatine kinases (EC 2.7.3) are homologous phosphagen kinases that catalyze the buffering of cellular ATP levels through phosphoryl transfer to/from their respective guanidino-containing substrates. The reaction is central to short-term temporal energy buffering (1, 2) as well as in spatial shuttling of energy from production to consumption sites (3-5). A wide array of endergonic processes is driven by nucleotide hydrolysis, from motion in molecular motors, active transport, and synthetic metabolism to signal transduction. Thus, the maintenance of a constant ATP/ADP ratio, displaced far from thermodynamic equilibrium in the face of high and variable rates of ATP turnover, is crucial for cellular homeostasis (2).Different organisms use different phosphagen substrates, usually only one and each with its own specific phosphagen kinase ( Fig. 1) (2). Lombricine kinase, as well as taurocyamine kinase and glycocyamine kinase, is found exclusively in annelids and allied groups (2). Phylogenetic analyses and studies of the intron/exon organization of the genes of these phosphagen kinases unique to annelids have shown that they are more closely related to creatine kinases (with which they share 50 -60% sequence identity) than typical monomeric arginine kinases such as that from the horseshoe crab (6) (40% sequence identity). Annelids are more diverse in their choice of phosphagen, and the substrate specificities of the corresponding kinases are often lower (6).Structural work has concentrated on the presumptive ancestral arginine kinase and the vertebrate creatine kinase (7, 8), but sequence alignment suggests that subunit fold, if not quaternary structure, is conserved across the fa...

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Section: Methodsmentioning

confidence: 95%

The Structure of Lombricine Kinase

Bush

Kirillova

Clark

et al. 2011

Journal of Biological Chemistry

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“…The resulting values for R -merge differed only minimally (<1%) and structure determination was subsequently attempted in P 2 1 2 1 2 1 , the space group found for the vast majority of crystals of A-form DNA 10mers with isolated 2′- O -modified residues investigated in our laboratory (20), and in space group P 4 2 2 1 2. Phasing was carried out by the molecular replacement method using the program AMORE (26) as part of the CCP4 suite of crystallographic programs (27). An A-form DNA decamer of the same sequence GCGTATAGCG (28) was used as the search model.…”

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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“…Data for cMutY K142A was collected at beamline 5.0.2 of the Advanced Light Source and processed with Mosflm and Scala. The structures were solved by molecular replacement using AmoRe with wild type cMutY as a search model (Protein Data Bank number 1MUY) (35). Refinement was done with CNS (36), and manual fitting was done with XFIT (37).…”

Section: Methodsmentioning

confidence: 99%

Reaction Intermediates in the Catalytic Mechanism of Escherichia coli MutY DNA Glycosylase

Manuel

Hitomi

Arvai

et al. 2004

Journal of Biological Chemistry

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The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary ␤-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-Å structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys 142 and Lys 20 . Analyses of the crystallographic structure also suggest a role for Glu 161 in the apurinic/ apyrimidinic lyase chemistry. The ␤-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes ␤ and ␤-␦ elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre-and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.Over the last 15 years, multiple laboratories have investigated the catalytic mechanism of DNA glycosylases that initiate the base excision repair (BER) 1 pathway (reviewed in Refs. 1-3). The elucidation of structure-activity relationships for DNA glycosylases and glycosylase/abasic site (AP) lyases has been facilitated both by solving the crystal structures and cocrystal complexes and analyses of chemical modification and trapping experiments (4 -17). These investigations have been successful in both localizing the active site pocket of these enzymes and identifying the key amino acids that participate in the catalytic events that lead to the excision of the inappropriate base. Determination of the chemical steps in the catalytic mechanism of DNA glycosylases is fundamental for understanding how organisms maintain their genome, despite the inevitable damage caused by exogenous and endogenous agents.DNA glycosylases with an associated AP lyase activity (␤-elimination) can be distinguished from DNA glycosylases without such activity, based on the identity of the nucleophile that attacks C1Ј of the deoxyribose sugar and whether the reaction proceeds through a covalent DNA-enzyme intermediate. DNA glycosylases that also catalyze a ␤-elimination reaction utilize a primary or secondary amine in the active site, whereas monofunctional glycosylases cleave the glycosyl bond via either the activation of a water molecule or a S N 1 attack (reviewed in Refs. 1 and 18). When a pr...

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[33] AMoRe: An automated molecular replacement program package

Cited by 552 publications

References 17 publications

The Structure of Lombricine Kinase

The Structure of Lombricine Kinase

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

Reaction Intermediates in the Catalytic Mechanism of Escherichia coli MutY DNA Glycosylase

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