Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER distance restraints.

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.

DOSE RECONSTRUCTION FROM ESR SIGNAL OF GAMMA-IRRADIATED SODA-LIME GLASS FOR TRIAGE APPLICATION.

In this work, we report some preliminary results regarding the analysis of electron spin resonance (ESR) response of soda-lime samples used for retrospective dosimetry. Six different soda-lime glass batches were evaluated after irradiation. We compared several dose reconstruction techniques: saturation method, subtraction method and g-effective, geff, approach. The differences were observed and discussed. ESR signal responses of soda-lime glass samples to different radiation doses for the triage application were investigated. Results confirmed that geff approach has potential for the identification and dosimetry of irradiated soda-lime glass samples using either additive dose method or only calibration curve.

A COMPARISON OF DIFFERENT SPECTRA DECONVOLUTION METHODS USED IN EPR DOSIMETRY WITH GORILLA® GLASSES.

Two different spectra deconvolution methods have been compared on samples of Gorilla® Glass (GG) irradiated in the dose range 0-20 Gy and measured with X-band EPR. The first method used a matrix deconvolution procedure using sample-specific sets of reference signals. The second method used a 'universal' set of eight reference signals (due to five electron centers, two hole centers and a background) to fit EPR spectra from any GG sample. Dose-responses curves were constructed for each individual reference signal. These were then used to test reconstruction of a laboratory-administered dose of 2 Gy. For the matrix method, the values of the reconstructed and nominal doses were within ± 20% after averaging measurements from three aliquots of each sample. For the universal method, the most promising results were obtained with E1, E4 and H1 signals. The fitting failed for one sample, due to dominance of the background signal.

Development of a novel mouth model as an alternative tool to test the effectiveness of an in vivo EPR dosimetry system.

In a large-scale radiation event, thousands may be exposed to unknown amounts of radiation, some of which may be life-threatening without immediate attention. In such situations, a method to quickly and reliably estimate dose would help medical responders triage victims to receive life-saving care. We developed such a method using electron paramagnetic resonance (EPR) to make in vivo measurements of the maxillary incisors. This report provides evidence that the use of in vitro studies can provide data that are fully representative of the measurements made in vivo. This is necessary because, in order to systematically test and improve the reliability and accuracy of the dose estimates made with our EPR dosimetry system, it is important to conduct controlled studies in vitro using irradiated human teeth. Therefore, it is imperative to validate whether our in vitro models adequately simulate the measurements made in vivo, which are intended to help guide decisions on triage after a radiation event. Using a healthy volunteer with a dentition gap that allows using a partial denture, human teeth were serially irradiated in vitro and then, using a partial denture, placed in the volunteer's mouth for measurements. We compared dose estimates made using in vivo measurements made in the volunteer's mouth to measurements made on the same teeth in our complex mouth model that simulates electromagnetic and anatomic properties of the mouth. Our results demonstrate that this mouth model can be used in in vitro studies to develop the system because these measurements appropriately model in vivo conditions.

Effect of the tooth surface water on the accuracy of dose reconstructions in the X-band in vivo EPR dosimetry.

The X-band in vivo EPR tooth dosimetry is promising as a tool for the initial triage after a large-scale radiation accident. The dielectric losses caused by water on the tooth surface (WTS) are one of the major sources of inaccuracies in this method. The effect was studied by theoretical simulation calculations and experiments with water films of various thicknesses on teeth. The results demonstrate the possibility of sufficiently accurate measurements of the radiation-induced signal of the tooth enamel provided that the thickness of the water film on the tooth is below 60 µm. The sensitivity of the cavity decreases with increasing thickness of the water layer. The interference of WTS can be diminished by normalization of the radiation-induced signal to the signal of a reference sample permanently present in the cavity.

Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.

While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available.

Full cycle rapid scan EPR deconvolution algorithm.

Rapid scan electron paramagnetic resonance (RS EPR) is a continuous-wave (CW) method that combines narrowband excitation and broadband detection. Sinusoidal magnetic field scans that span the entire EPR spectrum cause electron spin excitations twice during the scan period. Periodic transient RS signals are digitized and time-averaged. Deconvolution of absorption spectrum from the measured full-cycle signal is an ill-posed problem that does not have a stable solution because the magnetic field passes the same EPR line twice per sinusoidal scan during up- and down-field passages. As a result, RS signals consist of two contributions that need to be separated and postprocessed individually. Deconvolution of either of the contributions is a well-posed problem that has a stable solution. The current version of the RS EPR algorithm solves the separation problem by cutting the full-scan signal into two half-period pieces. This imposes a constraint on the experiment; the EPR signal must completely decay by the end of each half-scan in order to not be truncated. The constraint limits the maximum scan frequency and, therefore, the RS signal-to-noise gain. Faster scans permit the use of higher excitation powers without saturating the spin system, translating into a higher EPR sensitivity. A stable, full-scan algorithm is described in this paper that does not require truncation of the periodic response. This algorithm utilizes the additive property of linear systems: the response to a sum of two inputs is equal the sum of responses to each of the inputs separately. Based on this property, the mathematical model for CW RS EPR can be replaced by that of a sum of two independent full-cycle pulsed field-modulated experiments. In each of these experiments, the excitation power equals to zero during either up- or down-field scan. The full-cycle algorithm permits approaching the upper theoretical scan frequency limit; the transient spin system response must decay within the scan period. Separation of the interfering up- and down-field scan responses remains a challenge for reaching the full potential of this new method. For this reason, only a factor of two increase in the scan rate was achieved, in comparison with the standard half-scan RS EPR algorithm. It is important for practical use that faster scans not necessarily increase the signal bandwidth because acceleration of the Larmor frequency driven by the changing magnetic field changes its sign after passing the inflection points on the scan. The half-scan and full-scan algorithms are compared using a LiNC-BuO spin probe of known line-shape, demonstrating that the new method produces stable solutions when RS signals do not completely decay by the end of each half-scan.

Determination of the Average Native Background and the Light-Induced EPR Signals and their Variation in the Teeth Enamel Based on Large-Scale Survey of the Population.

The aim of the study is to determine the average intensity and variation of the native background signal amplitude (NSA) and of the solar light-induced signal amplitude (LSA) in electron paramagnetic resonance (EPR) spectra of tooth enamel for different kinds of teeth and different groups of people. These values are necessary for determination of the intensity of the radiation-induced signal amplitude (RSA) by subtraction of the expected NSA and LSA from the total signal amplitude measured in L-band for in vivo EPR dosimetry. Variation of these signals should be taken into account when estimating the uncertainty of the estimated RSA. A new analysis of several hundred EPR spectra that were measured earlier at X-band in a large-scale examination of the population of the Central Russia was performed. Based on this analysis, the average values and the variation (standard deviation, SD) of the amplitude of the NSA for the teeth from different positions, as well as LSA in outer enamel of the front teeth for different population groups, were determined. To convert data acquired at X-band to values corresponding to the conditions of measurement at L-band, the experimental dependencies of the intensities of the RSA, LSA and NSA on the m.w. power, measured at both X and L-band, were analysed. For the two central upper incisors, which are mainly used in in vivo dosimetry, the mean LSA annual rate induced only in the outer side enamel and its variation were obtained as 10 ± 2 (SD = 8) mGy y-1, the same for X- and L-bands (results are presented as the mean ± error of mean). Mean NSA in enamel and its variation for the upper incisors was calculated at 2.0 ± 0.2 (SD = 0.5) Gy, relative to the calibrated RSA dose-response to gamma radiation measured under non-power saturation conditions at X-band. Assuming the same value for L-band under non-power saturating conditions, then for in vivo measurements at L-band at 25 mW (power saturation conditions), a mean NSA and its variation correspond to 4.0 ± 0.4 (SD = 1.0) Gy.

ESR response of CFQ-Gd2O3 dosimeters to a mixed neutron-gamma field: Monte Carlo simulation.

Clear fused quartz (CFQ) may be considered a suitable material for electron and gamma dose measurements using electron spin resonance (ESR) technique. Research has been ongoing to optimize the neutron capture therapy (NCT) mechanism and its effects in cancer treatment. Neutron sources of the mixed neutron-gamma field are a challenge for this treatment method. A reliable dosimetric measurement and treatment should be able to determine various components of this mixed field. In this study, the ESR response of cylindrical and spherical shells of CFQ dosimeters, filled with Gd2O3, when exposed to a thermal neutron beam, has been investigated using Monte Carlo simulation. In order to maximize the ESR response, the dimensions of the outer and inner parts of the samples have been chosen as variables, and the amount of energy deposited in the samples has been determined. The optimum size of the samples has been determined, and the capability of discriminating gamma and neutron dose in a mixed neutron-gamma field regarding the CFQ-Gd2O3 dosimeter has also been widely studied.

Extended use of alanine irradiated in experimental reactor for combined gamma- and neutron-dose assessment by ESR spectroscopy and thermal neutron fluence assessment by measurement of (14)C by LSC.

Gamma- and neutron doses in an experimental reactor were measured using alanine/electron spin resonance (ESR) spectrometry. The absorbed dose in alanine was decomposed into contributions caused by gamma and neutron radiation using neutron kerma factors. To overcome a low sensitivity of the alanine/ESR response to thermal neutrons, a novel method has been proposed for the assessment of a thermal neutron flux using the (14)N(n,p) (14)C reaction on nitrogen present in alanine and subsequent measurement of (14)C by liquid scintillation counting (LSC).

Digital EPR with an arbitrary waveform generator and direct detection at the carrier frequency.

A digital EPR spectrometer was constructed by replacing the traditional bridge with an arbitrary waveform generator (AWG) to produce excitation patterns and a high-speed digitizer for direct detection of the spin system response at the carrier frequency. Digital down-conversion produced baseband signals in quadrature with very precise orthogonality. Real-time resonator tuning was performed by monitoring the Fourier transforms of signals reflected from the resonator during frequency sweeps generated by the AWG. The capabilities of the system were demonstrated by rapid magnetic field scans at 256 MHz carrier frequency, and FID and spin echo experiments at 1 and 10 GHz carrier frequencies. For the rapid scan experiments the leakage through a cross-loop resonator was compensated by adjusting the amplitude and phase of a sinusoid at the carrier frequency that was generated with another AWG channel.

Detection of undistorted continuous wave (CW) electron paramagnetic resonance (EPR) spectra with non-adiabatic rapid sweep (NARS) of the magnetic field.

A continuous wave (CW) electron paramagnetic resonance (EPR) spectrum is typically displayed as the first harmonic response to the application of 100 kHz magnetic field modulation, which is used to enhance sensitivity by reducing the level of 1/f noise. However, magnetic field modulation of any amplitude causes spectral broadening and sacrifices EPR spectral intensity by at least a factor of two. In the work presented here, a CW rapid-scan spectroscopic technique that avoids these compromises and also provides a means of avoiding 1/f noise is developed. This technique, termed non-adiabatic rapid sweep (NARS) EPR, consists of repetitively sweeping the polarizing magnetic field in a linear manner over a spectral fragment with a small coil at a repetition rate that is sufficiently high that receiver noise, microwave phase noise, and environmental microphonics, each of which has 1/f characteristics, are overcome. Nevertheless, the rate of sweep is sufficiently slow that adiabatic responses are avoided and the spin system is always close to thermal equilibrium. The repetitively acquired spectra from the spectral fragment are averaged. Under these conditions, undistorted pure absorption spectra are obtained without broadening or loss of signal intensity. A digital filter such as a moving average is applied to remove high frequency noise, which is approximately equivalent in bandwidth to use of an integrating time constant in conventional field modulation with lock-in detection. Nitroxide spectra at L- and X-band are presented.

Exact solution of the CPMG pulse sequence with phase variation down the echo train: application to R2 measurements.

An implicit exact algebraic solution of CPMG experiments is presented and applied to fit experiments. Approximate solutions are also employed to explore oscillations and effective decay rates of CPMG experiments. The simplest algebraic approximate solution has illustrated that measured intensities will oscillate in the conventional CPMG experiments and that using even echoes can suppress errors of measurements of R2 due to the imperfection of high-power pulses. To deal with low-power pulses with finite width, we adapt the effective field to calculate oscillations. An optimization model with the effective field approximation and dimensionless variables is proposed to quantify oscillations of measured intensities of CPMG experiments of different phases of the π pulses. We show, as was known using other methods, that repeating one group of four pulses with different phases in CPMG experiments, which we call phase variation, but others call phase alternation or phase cycling, can significantly smooth the dependence of measured intensities on frequency offset in the range of ±½Î³B1. In this paper, a second-order expression with respect to the ratio of frequency offset to π-pulse amplitude is developed to describe the effective R2 of CPMG experiments when using a group phase variation scheme. Experiments demonstrate that (1) the exact calculation of CPMG experiments can remarkably eliminate systematic errors in measured R2s due to the effects of frequency offset, even in the absence of phase variation; (2) CPMG experiments with group phase variation can substantially remove oscillations and effects of the field inhomogeneity; (3) the second-order expression of the effective decay rate with phase variation is able to provide reliable estimates of R2 when offsets are roughly within ±½Î³B1; and, most significantly, (4) the more sophisticated optimization model using an exact solution of the discretized CPMG experiment extends, to ±Î³B1, the range of offsets for which reliable estimates of R2 can be obtained when using the preferred phase variation scheme.

Reconstruction of the first-derivative EPR spectrum from multiple harmonics of the field-modulated continuous wave signal.

Selection of the amplitude of magnetic field modulation for continuous wave electron paramagnetic resonance (EPR) often is a trade-off between sensitivity and resolution. Increasing the modulation amplitude improves the signal-to-noise ratio, S/N, at the expense of broadening the signal. Combining information from multiple harmonics of the field-modulated signal is proposed as a method to obtain the first derivative spectrum with minimal broadening and improved signal-to-noise. The harmonics are obtained by digital phase-sensitive detection of the signal at the modulation frequency and its integer multiples. Reconstruction of the first-derivative EPR line is done in the Fourier conjugate domain where each harmonic can be represented as the product of the Fourier transform of the 1st derivative signal with an analytical function. The analytical function for each harmonic can be viewed as a filter. The Fourier transform of the 1st derivative spectrum can be calculated from all available harmonics by solving an optimization problem with the goal of maximizing the S/N. Inverse Fourier transformation of the result produces the 1st derivative EPR line in the magnetic field domain. The use of modulation amplitude greater than linewidth improves the S/N, but does not broaden the reconstructed spectrum. The method works for an arbitrary EPR line shape, but is limited to the case when magnetization instantaneously follows the modulation field, which is known as the adiabatic approximation.

Spinach--a software library for simulation of spin dynamics in large spin systems.

We introduce a software library incorporating our recent research into efficient simulation algorithms for large spin systems. Liouville space simulations (including symmetry, relaxation and chemical kinetics) of most liquid-state NMR experiments on 40+ spin systems can now be performed without effort on a desktop workstation. Much progress has also been made with improving the efficiency of ESR, solid state NMR and Spin Chemistry simulations. Spinach is available for download at http://spindynamics.org.

Multiple-stepped Zeeman field offset method applied in acquiring enhanced resolution spin-echo electron paramagnetic resonance images.

PURPOSE: Electron paramagnetic resonance (EPR) imaging techniques provide quantitative in vivo oxygen distribution images. Time-domain techniques including electron spin echo (ESE) imaging have been under study in recent years for their robustness and promising new features. One of the limitations of ESE imaging addressed here is the finite acquisition frequency bandwidth, which imposes limits on applied magnetic field gradients and the resulting image spatial resolution. In order to improve the image spatial resolution, we have extended the effective frequency bandwidth of the imaging system by acquiring projections at multiple Zeeman magnetic field offsets and combining them to restore complete projections obtained with more uniform frequency response, resulting in higher quality images. METHODS: In multiple-stepped magnetic field or multi-B scheme, every projection of the three dimensional object is acquired at different main or Zeeman magnetic field (B) offset values. The data from field offset steps are combined, normalizing to the imaging system frequency acquisition window function, a sensitivity profile, to restore the complete projection. A multipurpose pulse EPR imager and phantoms containing the same type of spin probe (OX063H) used in routine animal imaging were also used in this study. RESULTS: Using the multi-B method, we were able to acquire images of our phantoms with enhanced spatial resolution compared to the conventional ESE approach. Compared to standard single-B ESE images, the T2 resolutions of multi-B images were superior using a high spatial-resolution regime. Image artifacts present in high-gradient single-B ESE images are also substantially reduced using in the multi-B scheme. CONCLUSIONS: The multi-B method is less susceptible to instrumental limitations for larger gradient fields and acquiring images with higher spatial resolution better overall quality, without the need to alter the existing pulse ESE image acquisition hardware.

Experimental determination of the radial dose distribution in high gradient regions around 192Ir wires: comparison of electron paramagnetic resonance imaging, films, and Monte Carlo simulations.

PURPOSE: The experimental determination of doses at proximal distances from radioactive sources is difficult because of the steepness of the dose gradient. The goal of this study was to determine the relative radial dose distribution for a low dose rate 192Ir wire source using electron paramagnetic resonance imaging (EPRI) and to compare the results to those obtained using Gafchromic EBT film dosimetry and Monte Carlo (MC) simulations. METHODS: Lithium formate and ammonium formate were chosen as the EPR dosimetric materials and were used to form cylindrical phantoms. The dose distribution of the stable radiation-induced free radicals in the lithium formate and ammonium formate phantoms was assessed by EPRI. EBT films were also inserted inside in ammonium formate phantoms for comparison. MC simulation was performed using the MCNP4C2 software code. RESULTS: The radical signal in irradiated ammonium formate is contained in a single narrow EPR line, with an EPR peak-to-peak linewidth narrower than that of lithium formate (approximately 0.64 and 1.4 mT, respectively). The spatial resolution of EPR images was enhanced by a factor of 2.3 using ammonium formate compared to lithium formate because its linewidth is about 0.75 mT narrower than that of lithium formate. The EPRI results were consistent to within 1% with those of Gafchromic EBT films and MC simulations at distances from 1.0 to 2.9 mm. The radial dose values obtained by EPRI were about 4% lower at distances from 2.9 to 4.0 mm than those determined by MC simulation and EBT film dosimetry. CONCLUSIONS: Ammonium formate is a suitable material under certain conditions for use in brachytherapy dosimetry using EPRI. In this study, the authors demonstrated that the EPRI technique allows the estimation of the relative radial dose distribution at short distances for a 192Ir wire source.

Fitting of EPR spectra: the importance of a flexible bandwidth.

Utilizing a standard spin Hamiltonian for an S=32 spin system, we fit complete X-band powder EPR spectra of the Cr(oxalate)(3)(3-) anion diluted into K(3)[Co(oxalate)(3)]·3H(2)O. By using models for the bandshape and bandwidths of varying degree of flexibility, we show that the successful outcome of such a fitting endeavour very much depends on the used bandshape-bandwidth model. The best results are obtained when the bandwidth model incorporates anisotropic intrinsic bandwidths in addition to being able to account for inhomogeneous line broadening caused by distributions of the spin Hamiltonian parameters.

A signal-to-noise standard for pulsed EPR.

A 2 mm diameter by 10mm long cylinder of fused SiO2 (quartz) gamma-irradiated to 1 kGy with 60Co contains about 2x10(16) spins/cm3. It is proposed as a standard for monitoring signal-to-noise (S/N) performance of X-band pulsed EPR spectrometers. This sample yields S/N of about 25 on modern spin echo spectrometers, which permits measurement of both signal and noise under the same conditions with an 8-bit digitizer.