Pulsed Electron-Nuclear Double Resonance in the Fourier Regime.

Nuclear magnetic resonance (NMR) spectroscopy provides atomic-level molecular structural information. However, in molecules containing unpaired electron spins, NMR signals are difficult to measure directly. In such cases, data is obtained using the electron-nuclear double resonance (ENDOR) method, where nuclei are detected through their interaction with nearby unpaired electron spins. Unfortunately, electron spins spread the ENDOR signals, which challenges current acquisition techniques, often resulting in low spectral resolution that provides limited structural details. Here, we show that by using miniature microwave resonators to detect a small number of electron spins, integrated with miniature NMR coils, one can excite and detect a wide bandwidth of ENDOR data in a single pulse. This facilitates the measurement of ENDOR spectra with narrow lines spread over a large frequency range at much better spectral resolution than conventional approaches, which helps reveal details of the paramagnetic molecules' chemical structure that were not accessible before.

Nonuniformly sampled exclusively-13 C/15 N 4D solid-state NMR experiments: Assignment and characterization of IKe phage capsid.

An important step in the process of protein research by NMR is the assignment of chemical shifts. In the coat protein of IKe bacteriophage, there are 53 residues making up a long helix resulting in relatively high spectral ambiguity. Assignment thus requires the collection of a set of three-dimensional (3D) experiments and the preparation of sparsely labeled samples. Increasing the dimensionality can facilitate fast and reliable assignment of IKe and of larger proteins. Recent progress in nonuniform sampling techniques made the application of multidimensional NMR solid-state experiments beyond 3D more practical. 4D 1 H-detected experiments have been demonstrated in high-fields and at spinning speeds of 60 kHz and higher but are not practical at spinning speeds of 10-20 kHz for fully protonated proteins. Here, we demonstrate the applicability of a nonuniformly sampled 4D 13 C/15 N-only correlation experiment performed at a moderate field of 14.1 T, which can incorporate sufficiently long acquisition periods in all dimensions. We show how a single CANCOCX experiment, supported by several 2D carbon-based correlation experiments, is utilized for the assignment of heteronuclei in the coat protein of the IKe bacteriophage. One sparsely labeled sample was used to validate sidechain assignment of several hydrophobic-residue sidechains. A comparison to solution NMR studies of isolated IKe coat proteins embedded in micelles points to key residues involved in structural rearrangement of the capsid upon assembly of the virus. The benefits of 4D to a quicker assignment are discussed, and the method may prove useful for studying proteins at relatively low fields.

Cryo-electron microscopy structure of the filamentous bacteriophage IKe.

The filamentous bacteriophage IKe infects Escherichia coli cells bearing IncN pili. We report the cryo-electron microscopy structure of the micrometer-long IKe viral particle at a resolution of 3.4 Å. The major coat protein [protein 8 (p8)] consists of 47 residues that fold into a ∼68-Å-long helix. An atomic model of the coat protein was built. Five p8 helices in a horizontal layer form a pentamer, and symmetrically neighboring p8 layers form a right-handed helical cylinder having a rise per pentamer of 16.77 Å and a twist of 38.52°. The inner surface of the capsid cylinder is positively charged and has direct interactions with the encapsulated circular single-stranded DNA genome, which has an electron density consistent with an unusual left-handed helix structure. Similar to capsid structures of other filamentous viruses, strong capsid packing in the IKe particle is maintained by hydrophobic residues. Despite having a different length and large sequence differences from other filamentous phages, π-π interactions were found between Tyr9 of one p8 and Trp29 of a neighboring p8 in IKe that are similar to interactions observed in phage M13, suggesting that, despite sequence divergence, overall structural features are maintained.

Advanced surface resonators for electron spin resonance of single microcrystals.

Electron spin resonance (ESR) spectroscopy of paramagnetic species in single crystals is a powerful tool for characterizing the latter's magnetic interaction parameters in detail. Conventional ESR systems are optimized for millimeter-size samples and make use of cavities and resonators that accommodate tubes and capillaries in the range 1-5 mm. Unfortunately, in the case of many interesting materials such as enzymes and inorganic catalytic materials (e.g., zeolites), single crystals can only be obtained in micron-scale sizes (1-200 µm). To boost ESR sensitivity and to enable experiments on microcrystals, the ESR resonator needs to be adapted to the size and shape of these specific samples. Here, we present a unique family of miniature surface resonators, known as "ParPar" resonators, whose mode volume and shape are optimized for such micron-scale single crystals. This approach significantly improves upon the samples' filling factor and thus enables the measurement of much smaller crystals than was previously possible. We present here the design of such resonators with a typical mode dimension of 20-50 µm, as well as details about their fabrication and testing methods. The devices' resonant mode(s) are characterized by ESR microimaging and compared to the theoretical calculations. Moreover, experimental ESR spectra of single microcrystals with typical sizes of ∼25-50 µm are presented. The measured spin sensitivity for the 50-µm resonator at cryogenic temperatures of 50 K is found to be ∼1.8 × 106 spins/G √Hz for a Cu-doped single crystal sample that is representative of many biological samples of relevance.