Dataset Information

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

ABSTRACT: We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.

SUBMITTER: Mortensen KI

PROVIDER: S-EPMC4634324 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

Mortensen Kim I KI Sung Jongmin J Flyvbjerg Henrik H Spudich James A JA

Nature communications 20151016

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and or ...[more]

PMID: 26509412

Similar Datasets

Project description:Orthogonality metrics (OMs) for three and higher dimensional separations are proposed as extensions of previously developed OMs, which were used to evaluate the zone utilization of two-dimensional (2D) separations. These OMs include correlation coefficients, dimensionality, information theory metrics and convex-hull metrics. In a number of these cases, lower dimensional subspace metrics exist and can be readily calculated. The metrics are used to interpret previously generated experimental data. The experimental datasets are derived from Gilar's peptide data, now modified to be three dimensional (3D), and a comprehensive 3D chromatogram from Moore and Jorgenson. The Moore and Jorgenson chromatogram, which has 25 identifiable 3D volume elements or peaks, displayed good orthogonality values over all dimensions. However, OMs based on discretization of the 3D space changed substantially with changes in binning parameters. This example highlights the importance in higher dimensions of having an abundant number of retention times as data points, especially for methods that use discretization. The Gilar data, which in a previous study produced 21 2D datasets by the pairing of 7 one-dimensional separations, was reinterpreted to produce 35 3D datasets. These datasets show a number of interesting properties, one of which is that geometric and harmonic means of lower dimensional subspace (i.e., 2D) OMs correlate well with the higher dimensional (i.e., 3D) OMs. The space utilization of the Gilar 3D datasets was ranked using OMs, with the retention times of the datasets having the largest and smallest OMs presented as graphs. A discussion concerning the orthogonality of higher dimensional techniques is given with emphasis on molecular diversity in chromatographic separations. In the information theory work, an inconsistency is found in previous studies of orthogonality using the 2D metric often identified as %O. A new choice of metric is proposed, extended to higher dimensions, characterized by mixes of ordered and random retention times, and applied to the experimental datasets. In 2D, the new metric always equals or exceeds the original one. However, results from both the original and new methods are given.

Project description:BackgroundHemodynamic monitoring is imperative for patients with cardiogenic shock undergoing Intra-aortic Balloon Pump (IABP) therapy. Blood pressure monitoring encompasses non-invasive, invasive peripheral arterial pressure (IPAP), and invasive central aortic pressure (ICAP) methods. However, marked disparities exist between IPAP and ICAP. This study examined the discrepancies between IPAP and ICAP and their clinical significance.MethodsA retrospective analysis was conducted on cardiogenic shock patients who underwent IABP therapy and were admitted to the Coronary Care Unit (CCU) of a tertiary hospital in China from March 2017 to November 2022. The Bland-Altman plot illustrated the discrepancy between IPAP and ICAP. A clinically significant difference between ICAP and IPAP measurements was defined as ≥ 10 mmHg, which could necessitate alterations in blood pressure management according to current guidelines that recommend maintaining a mean arterial pressure (MAP) ≥ 70 mmHg.ResultsIn total, 162 patients were included in the final analysis. In patients without vasopressors, the difference between ICAP and IPAP was 5.73 mmHg (95% limits of agreement [LOA], -16.98 to 28.44), whereas, in patients with vasopressors, it was 4.36 mmHg (95% LOA, -17.31 to 26.03). ICAP measurements exceeded IPAP in patients undergoing IABP therapy. However, the difference was not statistically significant between the two groups. Multivariate logistic regression revealed that higher serum lactate levels (Odds ratio [OR], 1.14; 95% confidence interval [CI], 1.03-1.27; p = 0.013) and age ≥ 60 years (OR, 13.20; 95% CI, 1.50-115.51; p = 0.020) were associated with an increased likelihood of a clinically significant MAP discrepancy. Conversely, a history of coronary heart disease was associated with a decreased likelihood (OR, 0.34; 95% CI, 0.13-0.90; p = 0.031).ConclusionsNotable discrepancies between ICAP and IPAP measurements exist in cardiogenic shock patients undergoing IABP therapy. ICAP exceeds IPAP, and factors such as age ≥ 60 years, elevated lactic acid levels, and absence of coronary heart disease contribute to this discrepancy. Enhanced vigilance is warranted for these patients, and the consideration of peripheral invasive monitoring in conjunction with IABP therapy is advised.

Project description:BackgroundSeveral radiographic measurements of the humerus can be used to evaluate the treatment outcome of supracondylar fractures in children. Because of the cartilaginous nature of the immature elbow, interpretation of radiographs around this area is sometimes challenging and can be unreliable. This study was conducted to determine the inter-observer and intra-observer reliability of the six commonly used radiographic measurements of the distal humerus.MethodThe Baumann angle, humero-ulna angle, metaphyseal-diaphyseal angle, radial epiphyseal angle, shaft-condylar angle and lateral capitellohumeral angle of the humerus were measured by two observers on the radiographs of uninjured elbows from 58 children. The values between each measurement were compared and correlated using a Pearson coefficient of correlation to determine the inter-observer and intra-observer reliability.ResultsAll of the radiographic parameters showed excellent intra-observer reliability with the correlation coefficient values of the Baumann angle, humero-ulna angle, metaphyseal-diaphyseal angle, radial epiphyseal angle, shaft-condylar angle, lateral capitellohumeral angle as 0.945, 0.95, 0.909, 0.888, 0.961 and 0.975 (p < 0.001), respectively. The inter-observer reliability of the Baumann and humero-ulna angles were also found to be highly correlated at r = 0.843 (p < 0.001) and 0.878 (p < 0.001), respectively. The metaphyseal-diaphyseal angle had poor reliability with r = 0.136 (p = 0.291) while the radial epiphyseal angle, shaft-condylar angle, and lateral capitellohumeral angle demonstrated good reliability with r = 0.675 (p < 0.001), 0.747 (p < 0.001), and 0.686 (p < 0.001), respectively.ConclusionThe Baumann angle and humero-ulna angle measurements of distal humerus showed excellent inter- and intra-observer reliability. Both parameters represent repeatable and reliable methods for determining the outcome of supracondylar humeral fractures in pediatric population.

Project description:Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions.

Dataset Information

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

Publications

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets