analytical ultracentrifugation direct boundary modeling with sedfit

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Sedfit

This website provides information on Sedfit, a software for the analysis of analytical ultracentrifugation and other hydrodynamic data, written at the National Institutes of Health and distributed without charge for research use. The program can be downloaded from here, and it is also available directly from sedfitsedphat.nibib.nih.gov, or it can be sent as an email attachment from the author on request). This website was written by the author in his private capacity, and no official support or endorsement of NIH is intended and should be inferred. See the disclaimer.

As documentation please refer to the following books, which have specific information on SEDFIT and SEDPHAT functions as side-bars nested into the description of theory and experiment of analytical ultracentrifugation (AUC):

https://www.crcpress.com/Basic-Principles-of-Analytical-Ultracentrifugation/Schuck-Zhao-Brautigam-Ghirlando/p/book/9781498751155

https://www.crcpress.com/Sedimentation-Velocity-Analytical-Ultracentrifugation-Discrete-Species/Schuck/p/book/9781498768948

https://www.crcpress.com/Sedimentation-Velocity-Analytical-Ultracentrifugation-Interacting-Systems/Schuck-Zhao/p/book/9781138035287

A general introduction to the study of protein interactions by analytical ultracentrifugation can be found at the website of our lab at NIH (DMAS/LCIMB/NIBIB). This includes an introduction to the general principles of AUC and detailed experimental protocols.

The main purpose of this website is to provide additional information for using the software, such as an online help reference information, and background information (tutorials) on direct boundary modeling, FAQs, references, examples and more.

for upcoming workshops: see https://sedfitsedphat.nibib.nih.gov/workshop/default.aspx

Please join the Sedfit Users Group email list (reaches currently > 900 AUC users) for technical advice and discussions with other users on software issues and applications. You can also sign up to the SEDPHAT-L listserv for discussion of applications of SEDPHAT and questions of global analysis of ITC, AUC, DLS, SPR, and other biophysical techniques.

The main features of Sedfit include:

analysis of data from: sedimentation velocity, dynamic light scattering, and sedimentation equilibrium. Sedimentation velocity data can be acquired using absorbance or interference optics, in conventional loading configuration or synthetic boundary, analytical zone centrifugation, and different cell geometries

continuous size-distributions c(s) with many variants of prior knowledge for sedimentation velocity analysis with maximum entropy regularization

size-and-shape distributions c(s,M)

Bayesian incorporation of prior for enhanced resolution of distributions

Lamm equation models for: discrete non-interacting species, self-associating systems (1-2, 1-3, 1-2-4, 1-4-8, 1-m-n), non-ideal sedimentation --> see extension for global analysis of interacting systems in Sedphat

apparent sedimentation coefficient distribution ls-g*(s) and van Holde-Weischet analysis G(s) (both for absorbance and interference data)

all sedimentation velocity models for direct boundary modeling with algebraic noise elimination

corrections for water compressibility (and compressible organic solvents)

continuous size-distribution models for dynamic light scattering and sedimentation equilibrium

numerous statistical functions, loading options, and general purpose tools

extension to global analysis in Sedphat

Sedfit is closely related to Sedphat, which provides global modeling capability for both sedimentation equilibrium and sedimentation velocity data. It also can serve as a platform for the global analysis of a variety of isotherms from different biophysical disciplines.

NEW:

accounts for finite time of absorbance scanning

Extended multi-threading support for faster analysis on computers with multi-core processors

Bayesian prior knowledge for enhanced resolution of c(s) (links to paper and how to use in SEDFIT)

faster Lamm equation solutions (description)

new optimization tools: Marquardt-Levenberg and simulated annealing (how to use in SEDFIT)

real-time testing for attainment of sedimentation equilibrium (how to use in SEDFIT)

multi-thread support

new user interface (new color scheme, wizard messages, graphical editing of data points and scans, dragging limits with the mouse, quick change of cells, pre-set limits for automatic integration)

version 9.4: new specialized c(s) models (wormlike chain, floating vbar), extended calculator, display, and other utility functions (improved 6-channel data support)

version 9.3: size-and-shape distributions c(s,f) and c(s,M), and the scale-relationship free general c(s,*)

sounds, serialization of analysis (apply the same analysis automatically to data from several cells)

version 9.2: optional speeding up of c(s) analyses by slight radial pre-averaging of scans, faster Lamm equation solutions with permeable bottom model, c(s) model with user-defined M-s relationship, optional updating of display while fiitting, restoring previous c(s) analyses, loading data via drag-and-drop, importing SEDPHAT xp-files, and other new and improved utility functions.

new tutorials on solvent compressibility, and self-forming density gradients

new Sedphat hybrid bimodal c(s)-discrete species model combining 2 c(s) distributions with discrete species, for example allowing to link molar mass into integral multiples

version 8.9: Export data to Sedphat, and spawn Sedphat automatically with current data (and c(s) hybrid model).

new in version 8.8 (03/04): maintenance update with a few bug fixes and added flexibility

new in version 8.7 (09/03): sedimentation in inhomogeneous solvents, like sedimenting co-solutes and compressible solvents, including corrections for water compressibility; extended Monte-Carlo error analysis for weight-average s-values or concentrations of trace components; differential second moment method for sw; faster c(s) fitting; improved ellipsoid shape calculator; bimodal c(s) with two f/f0 parameters, back-transform for g(s*), several new utilities and bug fixes

new mode for REALTIME analysis

NEW REFERENCES:

Bayesian analysis of trace components (AAPS Journal 10 (2008) 481-493)

Bayesian prior knowledge (Biomacromolecules 8 (2007) 2011-2024)

new Lamm equation solutions (Computer Physics Communications in press)

size-and-shape distributions c(s,M) and scale-free general c(s,*) (Biophysical Journal 90 (2006) 4651-4661 full text )

interpreting c(s) for reacting systems: Biophysical Journal 89:651-666 and Biophysical Journal 89:619-634

tutorial review of analytical ultracentrifugation in protein science (Protein Science 11 (2002) 2067-2079)

new review of the strategies and numerical methods for sedimentation coefficient distributions in Methods in Enzymology (in press)

studies of strategies for using sedimentation velocity for protein self-association: Analytical Biochemistry 320:104-124

macromolecular sedimentation in the presence of sedimenting co-solutes (dynamic density gradients): Biophysical Chemistry 108:187-200

corrections for solvent compressibility Biophysical Chemistry 108:201-214

A snapshot from the direct boundary modeling of data from a sample of IgG, analyzed with a c(s) distribution of Lamm equation solutions illustrates the rich information contained in sedimentation velocity data.

This example also illustrates some of the main ideas of Sedfit: loading data from the entire sedimentation process, use of systematic noise decomposition (and subtraction), modeling with finite element solutions of the Lamm equation. If we expand the scale of the continuous sedimentation distribution c(s) with maximum entropy regularization shown above, it can be seen that the c(s) analysis reveals the presence of several oligomeric species and a smaller species:

Similarly, the analysis of data from a BSA sample

shows the presence of dimer and trimer in the sedimentation coefficient distribution c(s)

Transformation to a molar mass distribution (assuming that all species have a similar frictional ratio) is consistent with the oligomeric BSA species:

Analyses free of scale-relation ship assumptions of similar frictional ratios are available, such as the two-dimensional size-and-shape distribution c(s,f), c(s,M), and the general c(s,*).

Sedfit offers several different options for utilizing different prior knowledge on the sample under study (such as the number of discrete species, a self-association model, etc.). More information on these examples can be found in the step-by-step tutorial (BSA) and the example for using Sedfit (IgG).

For questions please contact the Sedfit Users Group email list .

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