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1.
Molecular replacement (MR) is widely used for addressing the phase problem in X-ray crystallography. Historically, crystallographers have had limited success using NMR structures as MR search models. Here, we report a comprehensive investigation of the utility of protein NMR ensembles as MR search models, using data for 25 pairs of X-ray and NMR structures solved and refined using modern NMR methods. Starting from NMR ensembles prepared by an improved protocol, FindCore, correct MR solutions were obtained for 22 targets. Based on these solutions, automatic model rebuilding could be done successfully. Rosetta refinement of NMR structures provided MR solutions for another two proteins. We also demonstrate that such properly prepared NMR ensembles and X-ray crystal structures have similar performance when used as MR search models for homologous structures, particularly for targets with sequence identity >40%. 相似文献
2.
DNA cloning without restriction enzyme and ligase 总被引:1,自引:0,他引:1
Tseng H 《BioTechniques》1999,27(6):1240-1244
One common problem in using the traditional DNA cloning procedure is that suitable natural restriction sites are often unavailable for a given task. Creating new restriction sites is often time consuming. Here, I describe a simple technique of producing "customized cohesive ends" by a combination of PCR primer design and lambda exonuclease digestion. These complementary cohesive ends can form hybrids to link two sequences. Because the overhangs created by lambda exonuclease are slightly longer than the complementary sequence, after hybrid formation, a stretch of single-strand gap remains, which then is repaired by Klenow (3'-->5' exo-) enzyme. The repair process also stabilizes the linkage. Because of the independence from natural or artificial restriction sites, this method allows rapid and precise insertion of one DNA fragment into another at virtually any position. It also simplifies the planning of a cloning strategy, increases recombinant frequency and is suitable for automation. 相似文献
3.
We review the role conformational ensembles can play in the analysis of biomolecular dynamics, molecular recognition, and
allostery. We introduce currently available methods for generating ensembles of biomolecules and illustrate their application
with relevant examples from the literature. We show how, for binding, conformational ensembles provide a way of distinguishing
the competing models of induced fit and conformational selection. For allostery we review the classic models and show how
conformational ensembles can play a role in unravelling the intricate pathways of communication that enable allostery to occur.
Finally, we discuss the limitations of conformational ensembles and highlight some potential applications for the future. 相似文献
4.
BACKGROUND: At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. RESULTS: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. CONCLUSIONS: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles. 相似文献
5.
It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade. Special emphasis is given to insights that run counter to some of the prevailing ideas that have emerged over the past 40 years of structural biology research. For instance, when proteins are viewed as conformational ensembles rather than as single structures, the commonly held notion of an allosteric pathway as an obligate series of individual structural distortions loses its meaning. Instead, allostery can result from energetic linkage between distal sites as one Boltzmann distribution of states transitions to another. Additionally, the emerging principles from this ensemble view of proteins have proven surprisingly useful in describing the role of intrinsic disorder in inter-domain communication, functional adaptation mediated by mutational control of fluctuations, and evolutionary conservation of the energetics of protein stability. 相似文献
6.
Lubomir Kovacik Jürgen M. Plitzko Monika Grote Rudolf Reichelt 《Journal of structural biology》2009,166(3):263-271
The three-dimensional structure of channels and bacula cavities in the wall of hazel pollen grains was investigated by automated electron tomography in order to explore their role in the release of allergen proteins from the pollen grains. 3D reconstructions of 100–150 nm thick resin-embedded sections, stabilized by thin platinum–carbon coating, revealed that the channels aimed directly towards the surface of the grain and that the bacula cavities were randomly sized and merged into larger ensembles. The number and the dimensions of the ensembles were quantitatively determined by neighboring voxel analysis on thresholded reconstructed volumes. To simulate the allergen release, allergen proteins were approximated by a hard sphere model of a diameter corresponding to the largest dimension of the known 3D structure of the major birch allergen, Bet v 1, whose amino acid sequence is highly similar to the amino acid sequence of the major hazel allergen, Cor a 1. The analysis of positions where the hard sphere fits into the resolved channels and bacula cavity structures revealed that unbound allergens could freely traverse through the channels and that the bacula cavities support the path of the allergens towards the surface of the grain. 相似文献
7.
Nucleotide sequence analysis of DNA. II. Complete nucleotide sequence of the cohesive ends of bacteriophage lambda DNA 总被引:43,自引:0,他引:43
At the ends of bacteriophage λ DNA, the 5′-terminated strands are 12 nucleotides longer than the 3′-terminated strands. The complete sequence of deoxynucleotides in both the protruding 5′-terminated single strands of λ DNA has been determined by partial repair and by complete repair followed by sequencing of isolated oligonucleotides. Starting from the 5′-end of the left-hand cohesive end, the 12 nucleotides are in the sequence dpGpGpGpCpGpGpCpGpApCpCpT. The sequence from the right-hand cohesive end is exactly complementary to that from the left-hand end. 相似文献
8.
Gell C Sabir T Westwood J Rashid A Smith DA Harris SA Stockley PG 《Journal of molecular biology》2008,384(1):264-278
We have examined the folding ensembles present in solution for a series of RNA oligonucleotides that encompass the replicase translational operator stem-loop of the RNA bacteriophage MS2. Single-molecule (SM) fluorescence assays suggest that these RNAs exist in solution as ensembles of differentially base-paired/base-stacked states at equilibrium. There are two distinct ensembles for the wild-type sequence, implying the existence of a significant free energy barrier between “folded” and “unfolded” ensembles. Experiments with sequence variants are consistent with an unfolding mechanism in which interruptions to base-paired duplexes, in this example by the single-stranded loop and a single-base bulge in the base-paired stem, as well as the free ends, act as nucleation points for unfolding. The switch between folded and unfolded ensembles is consistent with a transition that occurs when all base-pairing and/or base-stacking interactions that would orientate the legs of the RNA stem are broken. Strikingly, a U-to-C replacement of a residue in the loop, which creates a high-affinity form of the operator for coat protein binding, results in dramatically different (un)folding behaviour, revealing distinct subpopulations that are either stabilised or destabilised with respect to the wild-type sequence. This result suggests additional reasons for selection against the C-variant stem-loop in vivo and provides an explanation for the increased affinity. 相似文献
9.
Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with atopic dermatitis
Okamoto R Arikawa J Ishibashi M Kawashima M Takagi Y Imokawa G 《Journal of lipid research》2003,44(1):93-102
To clarify the functional relevance of sphingomyelin (SM) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [(14)C]acetic anhydride to yield [(14)C-C(2)]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD. 相似文献
10.
11.
Annamária F Ángyán Balázs Szappanos András Perczel Zoltán Gáspári 《BMC structural biology》2010,10(1):39
Background
In conjunction with the recognition of the functional role of internal dynamics of proteins at various timescales, there is an emerging use of dynamic structural ensembles instead of individual conformers. These ensembles are usually substantially more diverse than conventional NMR ensembles and eliminate the expectation that a single conformer should fulfill all NMR parameters originating from 1016 - 1017 molecules in the sample tube. Thus, the accuracy of dynamic conformational ensembles should be evaluated differently to that of single conformers. 相似文献12.
13.
Episodic-like memory is thought to be supported by attractor dynamics in the hippocampus. A possible neural substrate for this memory mechanism is rate remapping, in which the spatial map of place cells encodes contextual information through firing rate variability. To test whether memories are stored as multimodal attractors in populations of place cells, recent experiments morphed one familiar context into another while observing the responses of CA3 cell ensembles. Average population activity in CA3 was reported to transition gradually rather than abruptly from one familiar context to the next, suggesting a lack of attractive forces associated with the two stored representations. On the other hand, individual CA3 cells showed a mix of gradual and abrupt transitions at different points along the morph sequence, and some displayed hysteresis which is a signature of attractor dynamics. To understand whether these seemingly conflicting results are commensurate with attractor network theory, we developed a neural network model of the CA3 with attractors for both position and discrete contexts. We found that for memories stored in overlapping neural ensembles within a single spatial map, position-dependent context attractors made transitions at different points along the morph sequence. Smooth transition curves arose from averaging across the population, while a heterogeneous set of responses was observed on the single unit level. In contrast, orthogonal memories led to abrupt and coherent transitions on both population and single unit levels as experimentally observed when remapping between two independent spatial maps. Strong recurrent feedback entailed a hysteretic effect on the network which diminished with the amount of overlap in the stored memories. These results suggest that context-dependent memory can be supported by overlapping local attractors within a spatial map of CA3 place cells. Similar mechanisms for context-dependent memory may also be found in other regions of the cerebral cortex. 相似文献
14.
Preparation of multimilligram quantities of large, linear DNA molecules for structural studies 总被引:1,自引:0,他引:1
We describe a method for preparing large, linear DNA molecules in amounts that are suitable for structural studies. The procedure employs self-primed DNA amplification on a starting molecule that consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage lambda as well as endonuclease recognition sites. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. Endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of the DNA molecule of interest. Reliance on the cohesive end sequences to initiate self-primed amplification effectively enables the synthesis of DNA molecules of interest with minimal restriction on length and sequence. 相似文献
15.
16.
α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation. 相似文献
17.
Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing. 相似文献
18.
19.
Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment. 相似文献
20.
Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis we found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3′endo-anti gives a strong line at 666±2cm−1, O4′endo-anti at 682 cm−1, Cl′exo-anti at 673 cm−1, C2′endo-anti at 677 cm−1 and syn-forms around 625 cm−1. Using this characteristic line, we were able to obtain the local conformations of guanosine moieties in poly(dG-dC).Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. We found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base-long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA. 相似文献