Adam N Smith

Katharina Märker

Sabine Hediger

Gaël De Paëpe
email: gael.depaepe@cea.fr.

Natural Isotopic Abundance 13 C and 15 N Multidimensional Solid-State NMR Enabled by Dynamic Nuclear Polarization

Keywords: Magic angle spinning, Dynamic Nuclear Polarization, dipolar recoupling, sensitivity enhancement, correlation experiment, dipolar truncation

HAL is

The determination of molecular structure is essential to understand function. This canon is central to an array of fields, ranging from materials chemistry to structural biology. Over the years, magic angle spinning solid state NMR (MAS ssNMR) has proved invaluable in obtaining structural information with atomic resolution. A particular strength of the technique is that longrange order within the sample is not required (i.e. sans crystallization). As a result, heterogeneous molecular assemblies, such as amyloid fibrils, membrane proteins, heterogeneous catalysts, polymorphic pharmaceuticals, etc. are amenable to high-resolution structural studies by MAS ssNMR. [START_REF] Renault | Solid-State NMR Spectroscopy on Complex Biomolecules[END_REF][START_REF] Bryce | NMR Crystallography: Structure and Properties of Materials from Solid-State Nuclear Magnetic Resonance Observables[END_REF][START_REF] Harris | Applications of Solid-State NMR to Pharmaceutical Polymorphism and Related Matters[END_REF][START_REF] Zhang | In Situ Solid-State NMR for Heterogeneous Catalysis: A Joint Experimental and Theoretical Approach[END_REF] Despite the rich information content yielded from MAS ssNMR, the technique suffers from poor sensitivity. This lack of sensitivity is due to the small population difference between nuclear spin states at available magnetic field strengths. For example, only ~ 0.02 % of [START_REF] Renault | Solid-State NMR Spectroscopy on Complex Biomolecules[END_REF] H nuclei are polarized at 100 K and 23.5 T. The latter is considered under an ideal scenario, that is the polarization of a high gyromagnetic ratio nucleus (γ1H = 42.6 MHz/ T), at the highest commercially available NMR magnetic field ( 1 H resonant frequency = 1 GHz), and at cryogenic temperatures (100 K). Over the years, many advances in NMR were made to combat low sensitivity, including cryogenically cooled probes and preamplifiers, [START_REF] Kovacs | Cryogenically Cooled Probes -A Leap in NMR Technology[END_REF] efficient isotopic enrichment schemes, [START_REF] Hong | Selective and Extensive 13 C Labeling of a Membrane Protein for Solid-State NMR Investigations[END_REF][START_REF] Kwon | Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain[END_REF][START_REF] Higman | Assigning Large Proteins in the Solid State: A MAS NMR Resonance Assignment Strategy Using Selectively and Extensively 13 C-Labelled Proteins[END_REF][START_REF] Loquet | 13 C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies[END_REF][START_REF] Skrisovska | Recent Advances in Segmental Isotope Labeling of Proteins: NMR Applications to Large Proteins and Glycoproteins[END_REF] pulse sequence development, [START_REF] De Paëpe | Dipolar Recoupling in Magic Angle Spinning Solid-State Nuclear Magnetic Resonance[END_REF] and high magnetic field technology. [START_REF] Fu | Ultra-Wide Bore 900 MHz High-Resolution NMR at the National High Magnetic Field Laboratory[END_REF][START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] In addition, over the last two decades there has been a sensitivity revolution in solid-state NMR, namely the development of magic angle spinning dynamic nuclear polarization (MAS-DNP). [START_REF] Becerra | Dynamic Nuclear Polarization with a Cyclotron Resonance Maser at 5 T[END_REF][START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] In MAS-DNP experiments, the relatively larger polarization of electron spins in paramagnetic dopants is transferred to NMR active nuclei resulting in sensitivity gains that can yield several orders of magnitude in experimental time savings. [START_REF] Smith | Dynamic Nuclear Polarization as an Enabling Technology for Solid State Nuclear Magnetic Resonance Spectroscopy[END_REF][START_REF] Lee | Is Solid-State NMR Enhanced by Dynamic Nuclear Polarization? Solid State[END_REF] The substantial gain in sensitivity afforded by DNP has opened new experimental avenues in MAS ssNMR, for example, structural studies of proteins and materials at natural isotopic abundance (NA). Herein, we will discuss the advantages of MAS ssNMR experiments performed at NA and enabled by DNP, focusing on bio-organic applications that involve the detection of 1 H, 13 C, and [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N nuclei, and their use in structural measurements. Impressive results have also been obtained on other types of systems, involving the detection of low gamma nuclei at NA (e.g. [START_REF] Lee | Is Solid-State NMR Enhanced by Dynamic Nuclear Polarization? Solid State[END_REF] O, 43 Ca, [START_REF] Tycko | Amyloid Polymorphism: Structural Basis and Neurobiological Relevance[END_REF] Y, etc.), [START_REF] Blanc | Dynamic Nuclear Polarization Enhanced Natural abundance 17 O Spectroscopy[END_REF][START_REF] Blanc | Dynamic Nuclear Polarization NMR of Low-γ Nuclei: Structural Insights into Hydrated Yttrium-Doped BaZrO3[END_REF][START_REF] Perras | Characterizing Substrate-Surface Interactions on Alumina-Supported Metal Catalysts by Dynamic Nuclear Polarization-Enhanced Double-Resonance NMR Spectroscopy[END_REF][START_REF] Lee | Interfacial Ca2+ Environments in Nanocrystalline Apatites Revealed by Dynamic Nuclear Polarization Enhanced 43 Ca NMR Spectroscopy[END_REF] however, these studies are beyond the scope of this review.

-Motivation for NA ssNMR studies

The NA of NMR active nuclei varies largely throughout the periodic table. For example, [START_REF] Renault | Solid-State NMR Spectroscopy on Complex Biomolecules[END_REF] H is 99.9 % abundant, whereas [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C is only 1.1 % abundant. Table 1 lists the isotopic abundance for some of the more commonly observed NMR active nuclei, in addition the receptivity factor, a measure of how sensitive a particular nucleus is relative to 1 H is given. The high NA and large gyromagnetic ratio of 1 H (1H = 42.6 MHz/ T) makes it a logical (and popular) choice for NMR studies. However, in ssNMR, where anisotropic interactions such as dipolar couplings are not averaged by molecular tumbling, 1 H-detected NMR experiments can be problematic because of poor spectral resolution (resulting from the presence of large 1 H-1 H dipolar couplings). In contrast, heteronuclei (e.g. [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C, [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N, etc.) usually provide improved resolution thanks to larger spectral dispersion and narrower linewidths that can be obtained using efficient [START_REF] Renault | Solid-State NMR Spectroscopy on Complex Biomolecules[END_REF] H decoupling combined with MAS. To take advantage of the favorable characteristics of these heteronuclei with ssNMR, it is typically necessary to enrich the sample with the isotope of interest for the requisite sensitivity to perform advanced ssNMR experiments, such as recording multidimensional correlation spectra.

In fact, for many biomolecular systems, it has become relatively straightforward to isotopically enrich samples with [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C, [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N, and 2 H, both uniformly and sparsely with modern molecular biology techniques. [START_REF] Hong | Selective and Extensive 13 C Labeling of a Membrane Protein for Solid-State NMR Investigations[END_REF][START_REF] Kwon | Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain[END_REF][START_REF] Higman | Assigning Large Proteins in the Solid State: A MAS NMR Resonance Assignment Strategy Using Selectively and Extensively 13 C-Labelled Proteins[END_REF][START_REF] Loquet | 13 C Spin Dilution for Simplified and Complete Solid-State NMR Resonance Assignment of Insoluble Biological Assemblies[END_REF] However, for systems that are not amenable to those techniques, it is oftentimes cost prohibitive for isotopic enrichment due to the complex synthetic schemes used in their production, which makes it imperative to study such samples at their NA.

Also, in both material and biomolecular systems, the environment can influence the structural and functional characteristics of the sample. For example, the cellular milieu is composed of a complex mixture of small molecules, organelles, and proteins, all of which can have a dramatic influence on the structure of biomolecules. In addition, in-situ and operando studies of materials are becoming increasingly important in the development of catalysts, active pharmaceutical ingredients (APIs), functional polymers, carbohydrate-based materials, biomass treatment, batteries, etc. Therefore, there is a need to characterize these samples in, or derived from, their native environment. This frequently excludes isotopic enrichment protocols, necessitating the study of samples at NA.

Apart from the lower cost, higher availability, and relevance of NA samples, another exciting advantage of their use lies in the greatly simplified spin dynamics in dilute spin systems. For example, the observation and measurement of long-range distances (> 3 Å) is greatly facilitated as most spin pairs of interest are isolated. This means that MAS ssNMR experiments at NA can be advantageous, even when facile isotopic enrichment of samples is possible. This aspect of NA experiments will be explained in more detail in the following sections.

-Overcoming the sensitivity challenges of NA ssNMR by DNP

The low NA of NMR active nuclei presents a significant challenge with respect to experimental sensitivity, which can however be overcome by using MAS-DNP. Below, a brief description on MAS-DNP transfer mechanisms and the practical considerations of MAS-DNP experiments is

given.

An unpaired electron has a gyromagnetic ratio of ~ 28 GHz/ T, leading to a larger population difference (or polarization) between spin states compared to NMR active nuclei (γe/ γ1H ≈ 660). In DNP experiments, the aim is to transfer the larger polarization of an unpaired electron to nuclei resulting in a drastic increase in sensitivity. [START_REF] Overhauser | Polarization of Nuclei in Metals[END_REF][START_REF] Carver | Polarization of Nuclear Spins in Metals [15[END_REF] To date, the most efficient DNP transfer mechanism, at high-fields (≥ 5 T) and under MAS, is the cross-effect (CE). [START_REF] Mentink-Vigier | Efficient Cross-Effect Dynamic Nuclear Polarization without Depolarization in High-Resolution MAS NMR[END_REF] The CE occurs in a three-spin system of two electrons and one nucleus. The two electron spins are coupled by dipolar and exchange interactions, and inhomogenously broadened by a large g-anisotropy. The nucleus interacts with the electrons via hyperfine couplings. The energy levels from the resulting spin system are modulated by MAS and result in three distinct "rotor events" that mediate the generation of hyperpolarization of nuclei. [START_REF] Thurber | Theory for Cross Effect Dynamic Nuclear Polarization under Magic-Angle Spinning in Solid State Nuclear Magnetic Resonance: The Importance of Level Crossings[END_REF][START_REF] Mentink-Vigier | Fast Passage Dynamic Nuclear Polarization on Rotating Solids[END_REF][START_REF] Thurber | Perturbation of Nuclear Spin Polarizations in Solid State NMR of Nitroxide-Doped Samples by Magic-Angle Spinning without Microwaves[END_REF][START_REF] Mentink-Vigier | Nuclear Depolarization and Absolute Sensitivity in Magic-Angle Spinning Cross-Effect Dynamic Nuclear Polarization[END_REF] The first type of "rotor event" occurs when the resonant condition ωμw = ωe1 (or ωe2) is met and generates a large polarization difference between the two coupled electrons, where ωμw is the microwave irradiation frequency and ωe is an electron Larmor frequency. The second type of "rotor event" occurs when the CE condition |ωe1 -ωe2 |= ωn is met, where ωn is the nuclear Larmor frequency, yielding a transfer of the electron polarization difference to the nucleus. The third type of "rotor event" occurs when ωe1 = ωe2, termed a "dipolar event", and results in an exchange of electron polarization which should maintain the polarization difference between the two electrons to enable the buildup of nuclear hyperpolarization through successive CE events. Hyperpolarization is transferred from the hyperfine coupled nucleus to the bulk nuclei by nuclear spin diffusion.

In addition to the theoretical underpinnings of MAS-DNP transfer mechanisms, specialized instrumentation and sample preparation are required for efficient MAS-DNP to occur. The source of unpaired electrons -the polarizing agent -and its introduction into the sample is critical in MAS-DNP experiments. In general, suitable CE polarization agents consist of two radicals tethered together to form a biradical. [START_REF] Song | A Biradical Polarizing Agent for Dynamic Nuclear Polarization Experiments in Aqueous Media[END_REF] The use of biradicals ensures that the two unpaired electrons are strongly coupled by dipolar and exchange interactions. [START_REF] Hu | High-Frequency Dynamic Nuclear Polarization Using Biradicals: A Multifrequency EPR Lineshape Analysis[END_REF][START_REF] Mentink-Vigier | Computationally-Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family[END_REF] Oftentimes, the linker moiety is tuned to optimize these interactions. In addition, the linker can be functionalized to generate desired chemical properties (e.g. hydrophilicity) of the polarization agent or to target it to specific parts of a sample. [START_REF] Van Der Cruijsen | Biomolecular DNP-Supported NMR Spectroscopy Using Site-Directed Spin Labeling[END_REF][START_REF] Voinov | Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR[END_REF][START_REF] Salnikov | Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples[END_REF][START_REF] Rogawski | Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags[END_REF][START_REF] Smith | A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins[END_REF][START_REF] Smith | Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes[END_REF][START_REF] Fernández-De-Alba | Matrix-Free DNP-Enhanced NMR Spectroscopy of Liposomes Using a Lipid-Anchored Biradical[END_REF][START_REF] Albert | Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents[END_REF][START_REF] Takahashi | Solid-State NMR on Bacterial Cells: Selective Cell Wall Signal Enhancement and Resolution Improvement Using Dynamic Nuclear Polarization[END_REF] The polarization agent has to be homogeneously distributed in the sample to achieve efficient MAS-DNP. It is thus typically dissolved in a glass-forming mixture (containing glycerol or DMSO for instance) that can be used to suspend or impregnate the sample of interest. [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF][START_REF] Lesage | Surface Enhanced NMR Spectroscopy by Dynamic Nuclear Polarization[END_REF] Removal of the excess matrix can also be envisioned, where appropriate, either with the Matrix Free or Film Casting sample preparation approaches, as long as radical aggregation is avoided. [START_REF] Smith | A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins[END_REF][START_REF] Takahashi | Matrix-Free Dynamic Nuclear Polarization Enables Solid-State NMR 13 C-13 C Correlation Spectroscopy of Proteins at Natural Isotopic Abundance[END_REF][START_REF] Le | Optimizing Sample Preparation Methods for Dynamic Nuclear Polarization Solid-State NMR of Synthetic Polymers[END_REF] The instrumentation requirements for MAS-DNP are numerous, the primary components of a MAS-DNP system being a ssNMR magnet and spectrometer, a microwave source, and a cooling system coupled to a cryogenic MAS probe. To date, commercial MAS-DNP instrumentation is available at magnetic fields of 9.4, 14.1, 18.8, and 21.1 T. [START_REF] Rosay | Instrumentation for Solid-State Dynamic Nuclear Polarization with Magic Angle Spinning NMR[END_REF] The corresponding electron resonant frequencies (for a g  2 electron) for the latter magnetic fields are 263, 395, 527, and 591 GHz.

Currently, gyrotrons are the most utilized microwave sources capable of providing the required high-frequency microwave irradiation with sufficient power for CE MAS-DNP. Also, for favorable electron relaxation rates CE MAS-DNP is conducted at cryogenic temperatures. The sample is actively cooled and rotated at ~ 100 K with commercial MAS-DNP instrumentation. 44- 46 This is achieved by passing the N2 gas used to cool and spin the sample through pressurized heat exchangers that are submerged in liquid N2.

-MAS-DNP-assisted NMR crystallography

Structural studies of organic solids which are not suitable for single crystal X-ray diffraction can greatly profit from ssNMR. "NMR crystallography" studies have relied strongly on 1 H NMR (e.g.

1 H-X HETCOR spectra) and the comparison of experimental chemical shifts to those calculated by density functional theory (DFT) or other computational methods. [START_REF] Baias | De Novo Determination of the Crystal Structure of a Large Drug Molecule by Crystal Structure Prediction-Based Powder NMR Crystallography[END_REF][START_REF] Salager | Powder Crystallography by Combined Crystal Structure Prediction and High-resolution 1 H Solid-State NMR Spectroscopy[END_REF][START_REF] Paruzzo | Chemical Shifts in Molecular Solids by Machine Learning[END_REF] Through this comparison chemical structures can be validated from a given set of models. In general, 1 H-X HETCOR experiments are used because of the high sensitivity of 1 H compared to the lower-γ X nucleus.

From these experiments the chemical shifts of X-, and if resolution permits, 1 H-nuclei are measured for comparison to those calculated from candidate structures. In cases where greater spectral dispersion is required for the assignment of resonances 13 C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C correlation spectra can be acquired using INADEQUATE-type experiments. [START_REF] Lesage | Determination of through-Bond Carbon-Carbon Connectivities in Solid-State NMR Using the INADEAUATE Experiment[END_REF][START_REF] Lesage | Through-Bond Carbon-Carbon Connectivities in Disordered Solids by NMR[END_REF] These methods have been successfully used for structure determination on numerous systems and this type of approach has been expedited when used in combination with MAS-DNP techniques. [START_REF] Baias | De Novo Determination of the Crystal Structure of a Large Drug Molecule by Crystal Structure Prediction-Based Powder NMR Crystallography[END_REF][START_REF] Salager | Powder Crystallography by Combined Crystal Structure Prediction and High-resolution 1 H Solid-State NMR Spectroscopy[END_REF][START_REF] Andreas | Structure of Fully Protonated Proteins by Proton-Detected Magic-Angle Spinning NMR[END_REF][START_REF] Rossini | Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy[END_REF] Probing polymorphism using chemical shifts

Structural polymorphs occur in many solid-state systems and often have a dramatic impact on the function and properties of the material. Therefore, it is critical to be able to differentiate and characterize the different polymorphs within a given system. Chemical shifts report on the environment of their respective nuclei and are therefore particularly adept at characterizing structural polymorphs. APIs (active pharmaceutical ingredients) are a class of molecules that can have numerous polymorphic conformations, and it is necessary to characterize them before the pharmaceutical can be employed. In addition, the API is often diluted in excipients, this dilution makes them extremely difficult to characterize in-situ by conventional MAS ssNMR approaches, however, the added sensitivity of MAS-DNP enables in-situ studies of APIs at NA. [START_REF] Rossini | Dynamic Nuclear Polarization Enhanced NMR Spectroscopy for Pharmaceutical Formulations[END_REF] In one example, Ni Q.Z. et al. were able to obtain 1 H- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C and 1 H- [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N correlations of the API posaconazole in amorphous solid dispersions of vinyl acetate. [START_REF] Ni | In-Situ Characterization of Pharmaceutical Formulations by Dynamic Nuclear Polarization Enhanced MAS NMR[END_REF] Interestingly, in this study the MAS-DNP polarization agent was incorporated with the sample using spray drying or hot-melt extrusion, both of which are techniques to produce amorphous solid dispersions in the pharmaceutical industry. In a separate study by Pinon A.C. et al. of the API theophylline, MAS-DNP was used to characterize three polymorphs of the drug. In addition, they observed polymorphic transitions of the API that were induced by addition of the polarization agent by impregnation. [START_REF] Pinon | Polymorphs of Theophylline Characterized by DNP Enhanced Solid-State NMR[END_REF] However, they were able to devise alternative sample preparation techniques, such as grinding under an inert atmosphere, which prevented these induced polymorphic phase transitions.

DFT-free resonance assignment

With the limited 1 H resolution in ssNMR, ambiguities in the assignment of 1 H, [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C, and [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N resonances can easily occur in the approaches described above. Their assignment therefore necessarily relies on the comparison between experimentally measured chemical shifts and those computed from candidate structures. However, Märker et al. showed that even moderate (εon/off = 11) MAS-DNP enhancements are sufficient to record 2D 13 C-13 C and 13 C- [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N correlation spectra of a self-assembled deoxyguanosine derivative at NA. [START_REF] Märker | A New Tool for NMR Crystallography: Complete 13C/15N Assignment of Organic Molecules at Natural Isotopic Abundance Using DNP-Enhanced Solid-State NMR[END_REF] This enabled the full assignment of [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C and [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N resonances, which was not possible from proton-based correlation spectra alone due to the occurrence of two molecules in the asymmetric unit cell of the sample. In addition, the unambiguous assignment of chemical shifts did not rely on comparison to those computed by DFT.

In this instance, the enhanced sensitivity provided by MAS-DNP enabled 2D [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C and [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C-15 N through-space (dipolar) correlation experiments to be acquired. These dipolar-based experiments were necessary to obtain the atom connectivities because of the presence of interrupting nitrogen atoms in the deoxyguanosine ring structure, which prohibited the sole use of through-bond (J) correlation experiments.

-MAS-DNP-enabled NMR crystallography 13 C and 15 N spin dilution allows polarization transfer over long distance

The ability to use dipolar-based correlation experiments is important for probing proximities and thus to conduct resonance assignments (as explained above) but also raises the hope of accessing inter-nuclei distance information. Indeed, a mainstay of MAS ssNMR are the so-called dipolar recoupling experiments, where the dipolar coupling between nuclei, which is averaged by MAS, is reintroduced using dipolar recoupling pulse sequences. Because dipolar couplings are inversely proportional to the cube of the distance between the interacting nuclei, such experiments can be used to extract distance restraints. For instance, in the case of a doubly [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C labeled compound, a precise distance estimate can be obtained between the spin-pair based on the characteristic time course of the polarization buildup. [START_REF] De Paëpe | Dipolar Recoupling in Magic Angle Spinning Solid-State Nuclear Magnetic Resonance[END_REF] However, the situation is more complicated in the case of uniformly (or densely) [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C enriched samples, since a dense network of 13 C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C dipolar coupled nuclei is then created upon application of RF recoupling pulses. Such pulse sequences, called firstorder recoupling sequences, are part of a large toolbox that includes HORROR/DREAM, DRAWS, POSTC7, SPC5, SR26, CMRR/CMAR, S3 etc. [START_REF] Hohwy | Broadband Dipolar Recoupling in the Nuclear Magnetic Resonance of Rotating Solids: A Compensated C7 Pulse Sequence[END_REF][START_REF] Kristiansen | A Robust Pulse Sequence for the Determination of Small Homonuclear Dipolar Couplings in Magic-Angle Spinning NMR[END_REF][START_REF] Teymoori | Low-Power Broadband Homonuclear Dipolar Recoupling without Decoupling: Double-quantum13C NMR Correlations at Very Fast Magic-Angle Spinning[END_REF][START_REF] Teymoori | Low-Power Broadband Homonuclear Dipolar Recoupling in MAS NMR by Two-Fold Symmetry Pulse Schemes for Magnetization Transfers and Double-Quantum Excitation[END_REF][START_REF] Gregory | Windowless Dipolar Recoupling: The Detection of Weak Dipolar Couplings between Spin 1 2 Nuclei with Large Chemical Shift Anisotropies[END_REF][START_REF] Hohwy | Fivefold Symmetric Homonuclear Dipolar Recoupling in Rotating Solids: Application to Double Quantum Spectroscopy[END_REF][START_REF] De Paëpe | Broadband Homonuclear Correlation Spectroscopy at High Magnetic Fields and MAS Frequencies[END_REF][START_REF] De Paëpe | Spin Dynamics in the Modulation Frame: Application to Homonuclear Recoupling in Magic Angle Spinning Solid-State NMR[END_REF][START_REF] Nielsen | Double-Quantum Homonuclear Rotary Resonance: Efficient Dipolar Recovery in Magic-Angle Spinning Nuclear Magnetic Resonance[END_REF][START_REF] Verel | A Homonuclear Spin-Pair Filter for Solid-State NMR Based on Adiabatic-Passage Techniques[END_REF] One of the consequences of the complex multi-spin dynamics is that the extraction of precise long distance information is becoming impossible because of a phenomenon called dipolar truncation. [START_REF] De Paëpe | Dipolar Recoupling in Magic Angle Spinning Solid-State Nuclear Magnetic Resonance[END_REF][START_REF] Costa | Peptides, and Alzheimer's Disease: Solid-State Nuclear Magnetic Resonance Investigations of Amyloid Peptide Conformation[END_REF] This is illustrated in Figure 1a, which shows that polarization transfer to a distant nucleus (4.4 Å here) is strongly attenuated in the presence of a larger dipolar coupling, i.e. another nucleus at a shorter distance (1.5 Å). Such attenuation prevents the detection and the extraction of precise long distance information which would be valuable for structural studies. [START_REF] Costa | Peptides, and Alzheimer's Disease: Solid-State Nuclear Magnetic Resonance Investigations of Amyloid Peptide Conformation[END_REF][START_REF] De Paëpe | Proton Assisted Recoupling and Protein Structure Determination[END_REF] As a result, 2D [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C correlation experiments performed on uniformly [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C labeled compounds (Figure 2) only contain cross-peaks that correspond to short distance correlations (one-bond transfer). Similar behavior can be expected for other densely coupled homonuclear networks, including 1 H-1 H spin systems under ultrafast-MAS. 70 The dipolar truncation effect has long been a major bottleneck for structure determination of isotopically labeled biomolecular systems. As seen above, this effect is very intense for first-order homonuclear recoupling pulse sequences. It is however also present in heteronuclear cases if the dipolar recoupled terms do not commute with each other, like for instance in cross polarization (CP) based pulse sequences. This is not the case for REDOR and TEDOR heteronuclear recoupling sequences, which explains why these sequences were successfully used to extract precise distance observed in the U- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C, [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N enriched histidine due to the effects of dipolar truncation. In addition, in the U- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C, [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N sample at the 4 ms mixing time the spectrum is complicated by the presence of correlations resulting from indirect transfers (green diamonds). [START_REF] Brinkmann | Synchronous Helical Pulse Sequences in Magic-Angle Spinning Nuclear Magnetic Resonance: Double Quantum Recoupling of Multiple-Spin Systems[END_REF] Asterisks mark spinning sidebands. The difference in assignment of the aromatic carbons is a result of different histidine tautomers between the two samples. [START_REF] Sudmeier | Identification of Histidine Tautomers in Proteins by 2D 1H/13Cδ2 One-Bond Correlated NMR[END_REF] The spectra in b) were recorded using MAS-DNP.

information in [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N/ [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C labeled biomolecular systems. Still, even in these cases the presence of multiple spins complicates the analysis of experimental data, as shown for TEDOR experiments for example. [START_REF] Jaroniec | 3D TEDOR NMR Experiments for the Simultaneous Measurement of Multiple Carbon-Nitrogen Distances in Uniformly 13C,15N-Labeled Solids[END_REF] To overcome the limitations described above, recoupling methods were developed with the goal to reduce dipolar truncation effects. Among them stand the Third Spin Assisted Recoupling (TSAR) methods, in which the polarization transfer between two spins X1 and X2 involves the couplings to an assisting nucleus, typically 1 H. More precisely, the associated pulse sequence relies on second-order cross-terms arising from 1 H-X1 and 1 H-X2 (where X = 13 C, [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N, …) interactions. [START_REF] De Paëpe | Spin Dynamics in the Modulation Frame: Application to Homonuclear Recoupling in Magic Angle Spinning Solid-State NMR[END_REF][START_REF] Lewandowski | Proton Assisted Insensitive Nuclei Cross Polarization[END_REF][START_REF] Paëpe | Heteronuclear Proton Assisted Recoupling[END_REF] The benefit of this approach is that long-distance correlations can be observed even in presence of strong dipolar couplings. However, it is important to stress that precise X1-X2 distance fitting remains challenging, principally because the polarization transfer does not directly depend upon the X1-X2 couplings, but rather upon the positioning of the surrounding 1 H spins. [START_REF] De Paëpe | Proton Assisted Recoupling and Protein Structure Determination[END_REF][START_REF] Paëpe | Heteronuclear Proton Assisted Recoupling[END_REF] Nevertheless, this approach can be used to estimate upper and/or lower bound distances and has been shown to provide very useful distance constraints for protein structure determination in ssNMR. [START_REF] De Paëpe | Proton Assisted Recoupling and Protein Structure Determination[END_REF][START_REF] Bertini | High-Resolution Solid-State NMR Structure of a 17 . 6 kDa Protein[END_REF] The problem associated with dipolar truncation can be foiled by working with dilute spins, as is the case with [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C and 15 N spins at their NA. For example, [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C is 1.11 % abundant and as a result every particular [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C spin pair is only 0.01 % abundant. Therefore, the probability of a 13 C-13 C spin-pair encountering a third perturbing [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C nucleus that would contribute to dipolar truncation is exceedingly low. [START_REF] Märker | Welcoming Natural Isotopic Abundance in Solid-State NMR: Probing Π-Stacking and Supramolecular Structure of Organic Nanoassemblies Using DNP[END_REF][START_REF] Takahashi | Towards Structure Determination of Self-Assembled Peptides Using Dynamic Nuclear Polarization Enhanced Solid-State NMR Spectroscopy[END_REF][START_REF] Takahashi | Rapid Natural-Abundance 2D 13 C-13 C Correlation Spectroscopy Using Dynamic Nuclear Polarization Enhanced Solid-State NMR and Matrix-Free Sample Preparation[END_REF] Because of this spin dilution effect at NA, first-order homonuclear dipolar recoupling sequences can now be used to detect long-distance polarization transfer once the sensitivity limitation is negated by the use of MAS-DNP. [START_REF] Takahashi | Towards Structure Determination of Self-Assembled Peptides Using Dynamic Nuclear Polarization Enhanced Solid-State NMR Spectroscopy[END_REF][START_REF] Takahashi | Rapid Natural-Abundance 2D 13 C-13 C Correlation Spectroscopy Using Dynamic Nuclear Polarization Enhanced Solid-State NMR and Matrix-Free Sample Preparation[END_REF] This has been demonstrated in 2012 by Takahashi et al., [START_REF] Takahashi | Rapid Natural-Abundance 2D 13 C-13 C Correlation Spectroscopy Using Dynamic Nuclear Polarization Enhanced Solid-State NMR and Matrix-Free Sample Preparation[END_REF] and later used to detect both intra and intermolecular contacts in selfassembled peptides, [START_REF] Takahashi | Towards Structure Determination of Self-Assembled Peptides Using Dynamic Nuclear Polarization Enhanced Solid-State NMR Spectroscopy[END_REF] and to identify the correct polymorph of the pharmaceutical compound theophylline, as reported by Mollica et al., thanks to the use of dipolar buildups. 80

Spin dilution grants access to precise long-range measurements

Later in 2017, Märker et al. presented the first detailed analysis of polarization buildup curves from [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C-13 C dipolar recoupling experiments at NA. [START_REF] Märker | Welcoming Natural Isotopic Abundance in Solid-State NMR: Probing Π-Stacking and Supramolecular Structure of Organic Nanoassemblies Using DNP[END_REF] It was shown that each buildup curve represents a superposition of buildup curves from several distance contributions, many of them being intermolecular, and that they are sensitive to distances up to ~7 Å. The simulation of spin pairs was sufficient to model these buildup curves and excellent agreement was obtained between the experimental buildup curves and simulated curves based on the crystal structure. It is also possible to see several distances contribute to the same buildup curve when a given spin pair occurs at different distances. This can for instance be the case in a solid composed of small organic molecules where nucleus A can "see" nucleus B in the same molecule (intramolecular), but also in adjacent molecules (intermolecular). Therefore, experimental polarization buildup curves are often composed of a superposition of buildup curves corresponding to the different distances for a given spin-pair as illustrated in Figure 4. Multiple distances can in principle be extracted from a given build up curve. However, an upper cutoff of the longest observable distance still exists, which is given by the efficiency of the dipolar recoupling sequence employed, but also by the probability of encountering a third truncating spin. This probability increases as the observation radius around the starting nucleus is increased and will eventually be 100 %, meaning that, statistically, no polarization transfer beyond this distance will be possible.

This cutoff distance was estimated to lie between 7 and 8 Å in an organic solid with densely packed molecules. 77 Another benefit of recording polarization buildup curves at NA is that their analysis by numerical simulations (e.g. with SPINEVOLUTION, SIMPSON, etc… [START_REF] Veshtort | A Powerful Tool for the Simulation of Solid and Liquid State NMR Experiments[END_REF][START_REF] Bak | A General Simulation Program for Solid-State NMR Spectroscopy[END_REF] ) is considerably simplified compared to isotopically enriched samples. It is sufficient to consider only two interacting nuclei in each simulation and to sum up such spin-pair simulations as needed. This results in very fast calculation times, especially compared to the more time-consuming simulation of multi-spin systems with various interacting nuclei and geometries. 81,83 13 C spin dilution requires the use of DQ filtered experiments or selective spin flip.

While in general the same pulse sequences can be used with and without DNP, dipolar recoupling experiments between dilute nuclei pose special challenges. These arise because of the even lower abundance of the required spin pairs as most nuclei are not dipolar coupled to another nucleus of interest. Signals arising from spin pairs would therefore be buried under the much more intense signals originating from uncoupled spins. While this problem is naturally circumvented in heteronuclear correlation experiments, homonuclear correlations of dilute spins are greatly improved by using DQ filtration. [START_REF] Wokaun | Selective Detection of Multiple Quantum Transitions in NMR[END_REF][START_REF] Bax | An NMR Technique for Tracing Out the Carbon Skeleton of an Organic Molecule[END_REF] DQ-filtered experiments employ pulse sequences to create DQ coherences and exclusively select these coherences with a suitable phase cycle. As a DQ coherence can only be created between dipolar coupled spins, DQ filtration efficiently suppresses all signals from uncoupled spins. For such experiments, it should be kept in mind that the DQ filter removes half the signal, leading to a loss in sensitivity. [START_REF] Nielsen | Double-Quantum Homonuclear Rotary Resonance: Efficient Dipolar Recovery in Magic-Angle Spinning Nuclear Magnetic Resonance[END_REF] Another way of suppressing the signal of uncoupled spins can be the use of selective pulses as demonstrated by Märker et al. for recoupling of [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C nuclei at NA. [START_REF] Märker | Welcoming Natural Isotopic Abundance in Solid-State NMR: Probing Π-Stacking and Supramolecular Structure of Organic Nanoassemblies Using DNP[END_REF] By selectively exciting a subset of resonances and subsequent application of a recoupling sequence, any signals building up on other resonances have to originate from the initially excited spin(s). [START_REF] Märker | Welcoming Natural Isotopic Abundance in Solid-State NMR: Probing Π-Stacking and Supramolecular Structure of Organic Nanoassemblies Using DNP[END_REF] Such an experiment can be run without DQ filtration and collected as a series of 1D spectra, in contrast to the traditional approach that requires a set of 2D DQ/SQ correlation spectra to be recorded. This 1D method can therefore be very fast and attractive, but crucially depends on the applicability of selective pulses,

i.e. it requires sufficient spectral dispersion of resonances.

When measuring long distances, i.e. small dipolar couplings, long recoupling times are required and the ideal recoupling sequence needs to be very robust with respect to interfering interactions such as chemical shift anisotropies and radio frequency pulse imperfections. Supercycled pulse sequences are usually best-suited for this purpose. [START_REF] Kristiansen | Theory and Applications of Supercycled Symmetry-Based Recoupling Sequences in Solid-State Nuclear Magnetic Resonance[END_REF] Efficient pulse sequences for homonuclear (DQ) recoupling are, for example, SR26 and S3, [START_REF] Kristiansen | A Robust Pulse Sequence for the Determination of Small Homonuclear Dipolar Couplings in Magic-Angle Spinning NMR[END_REF][START_REF] Teymoori | Low-Power Broadband Homonuclear Dipolar Recoupling without Decoupling: Double-quantum13C NMR Correlations at Very Fast Magic-Angle Spinning[END_REF][START_REF] Teymoori | Low-Power Broadband Homonuclear Dipolar Recoupling in MAS NMR by Two-Fold Symmetry Pulse Schemes for Magnetization Transfers and Double-Quantum Excitation[END_REF] whereas the z-filtered TEDOR sequence is a good option for heteronuclear recoupling. [START_REF] Jaroniec | 3D TEDOR NMR Experiments for the Simultaneous Measurement of Multiple Carbon-Nitrogen Distances in Uniformly 13C,15N-Labeled Solids[END_REF]

-Structural biology at NA

The structural characterization of biomolecules by ssNMR also benefits from experiments at NA, as long-range distance constraints again convey the most valuable structural information.

Therefore, the reduced effects of dipolar truncation (discussed above) are also key in the characterization of biomolecules. An initial application of this approach was shown by Takahashi et al. where long-range correlations were observed for a self-assembled peptide and a globular protein using DQ/SQ 13 C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C correlation experiments at NA, enabled by MAS-DNP. [START_REF] Takahashi | Matrix-Free Dynamic Nuclear Polarization Enables Solid-State NMR 13 C-13 C Correlation Spectroscopy of Proteins at Natural Isotopic Abundance[END_REF][START_REF] Takahashi | Towards Structure Determination of Self-Assembled Peptides Using Dynamic Nuclear Polarization Enhanced Solid-State NMR Spectroscopy[END_REF] Previously, Zhou et al. showed that [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C-1 H and [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N-1 H correlation spectra of proteins could be obtained at NA, however, this was accomplished without MAS-DNP and therefore necessarily relied on the use of 1 H-detection schemes. [START_REF] Zhou | Proton-Detected Solid-State NMR Spectroscopy of Natural-Abundance Peptide and Protein Pharmaceuticals[END_REF] In addition to avoiding the deleterious effects of dipolar truncation, it is also advantageous to study biomolecules at NA because it allows studying samples which are derived from sources that are difficult or impossible to isotopically enrich. One such example are amyloid fibrils, which are recognized for their role in numerous neurodegenerative diseases. It is known that the conditions under which protein monomers undergo aggregation can affect the conformation of the resulting fibril, [START_REF] Van Der Wel | Insights into Protein Misfolding and Aggregation Enabled by Solid-State NMR Spectroscopy[END_REF][START_REF] Tycko | Amyloid Polymorphism: Structural Basis and Neurobiological Relevance[END_REF][START_REF] Frederick | Sensitivity-Enhanced NMR Reveals Alterations in Protein Structure by Cellular Milieus[END_REF] and it has been shown that different polymorphs can display varying levels of neurotoxicity. [START_REF] Lin | Fibril Polymorphism Affects Immobilized Non-Amyloid Flanking Domains of Huntingtin exon1 rather than Its Polyglutamine Core[END_REF] Therefore, it is imperative to characterize the structure of protein aggregates that are formed under native conditions. One route in achieving this goal is to study protein aggregates derived from patients or animal models, which necessitate that the aggregates are at NA. The prospects of this approach have been shown by Smith et al. who obtained [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C DQ/SQ and 13 C- [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N correlation spectra for NA poly-glutamine (polyQ) fibrils which are the hallmark of Huntington's Disease. [START_REF] Smith | Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance[END_REF] These experiments demonstrated that a fingerprint, via [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C and [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF] N chemical shift assignment (Figure 5), of the amyloid core could be obtained at NA for protein fibrils. In addition, long-range distance restraints were obtained and aided in the characterization of the β-strand arrangement in the polyQ amyloid core. These types of measurements would not have been possible in uniformly [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C enriched protein samples due to the effects of dipolar truncation. Another example of the utility of NA studies of biomolecules was shown by Sinh S. et al. [START_REF] Singh | Direct Evidence of Imino Acid-Aromatic Interactions in Native Collagen Protein by DNP-Enhanced Solid-State NMR Spectroscopy[END_REF] who used MAS-DNP to investigate native bone collagen at NA. While collagen can be produced invitro, it is important to study it from native sources due to its numerous interactions with noncollagenous proteins that can impact its structure. In their study, CH/ interactions between the protons from the C and C of proline and hydroxyproline and the ring carbons of aromatic residues were observed via 1 H- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C correlation experiments (Figure 6). Understanding these interactions is important because it is postulated that intermolecular interactions between aromatic residues and hydroxyproline and proline residues promote self-aggregation in collagen triple helices. These correlations are particularly difficult to observe if no MAS-DNP is employed due to the low occurrence (~ 1 %) of aromatic residues in collagen.

-Outlook

NA ssNMR experiments are challenging, however, with the impressive gains in sensitivity realized with MAS-DNP they are now achievable. Herein, we presented the advantages of NA ssNMR and some recent applications of the technique. Despite the significant progress made in this branch of MAS-DNP, there are still many advances to be made in the coming years, specifically, with further gains in MAS-DNP efficiency. Larger MAS-DNP enhancements will allow even more challenging samples to be studied and even more advanced/complicated NMR experiments to be performed at NA. Improvements in MAS-DNP efficiency will be ascertained through concerted efforts primarily in hardware and polarization agent development and improved computational modeling.

A number of research groups are developing He based MAS instrumentation and have been able

to perform MAS-DNP experiments at temperatures much lower than 100 K. [START_REF] Hall | Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution[END_REF][START_REF] Bouleau | Pushing NMR Sensitivity Limits Using Dynamic Nuclear Polarization with Closed-Loop Cryogenic Helium Sample Spinning[END_REF][START_REF] Thurber | Low-Temperature Dynamic Nuclear Polarization with Helium-Cooled Samples and Nitrogen-Driven Magic-Angle Spinning[END_REF][START_REF] Sesti | Electron Decoupling with Cross Polarization and Dynamic Nuclear Polarization below 6 K[END_REF][START_REF] Matsuki | Helium-Cooling and -Spinning Dynamic Nuclear Polarization for Sensitivity-Enhanced Solid-State NMR at 14 T and 30[END_REF] He MAS-DNP instrumentation is advantageous for numerous reasons, including, more favorable electron relaxation rates which should yield larger enhancements. Also, the fluid dynamic properties of He are better suited for fast MAS at similar temperatures compared to N2 gas. [START_REF] Bouleau | Pushing NMR Sensitivity Limits Using Dynamic Nuclear Polarization with Closed-Loop Cryogenic Helium Sample Spinning[END_REF][START_REF] Lee | Ultra-Low Temperature MAS-DNP[END_REF] Currently, commercial MAS-DNP instrumentation uses N2 for cooling and MAS, using He should allow rotors to be spun at rates comparable to conventional ssNMR (e.g. ~ 25 kHz for 3.2 mm rotors and ~ 60 kHz for 1.3 mm rotors). Faster MAS-DNP will expand the repertoire of pulse sequences that can be used at NA, for example, broadband dipolar recoupling sequences, and the use of low power or no 1 H decoupling. [START_REF] De Paëpe | Dipolar Recoupling in Magic Angle Spinning Solid-State Nuclear Magnetic Resonance[END_REF] Improved polarization agents will also lead to more efficient MAS-DNP. Recently, a new class of nitroxide biradicals were introduced, the AsymPol family, which were designed with the aid of computational methods. [START_REF] Mentink-Vigier | Computationally-Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family[END_REF] This represents a large step in the development of polarization agents that had solely relied on empirical evaluation in new radical design. The improved theoretical understanding of MAS-DNP mechanisms and the use of computational modeling of MAS-DNP processes will no doubt lead to further improvements in polarization agents. In addition to a new class of nitroxide biradicals, mixed biradicals also show promise for performing NA MAS-DNP at high-field (i.e. > 9.4 T). The combination of a narrow line radical (e.g. trityl or BDPA) and a wide line radical (e.g. nitroxide) have been used to perform DQ/SQ 13 C- [START_REF] Gan | NMR Spectroscopy up to 35.2 T Using a Series-Connected Hybrid Magnet[END_REF] C NA experiments on cellulose at 18.8 T (Figure 7); this was the first demonstration of NA MAS-DNP at high-field. [START_REF] Mentink-Vigier | Efficient Cross-Effect Dynamic Nuclear Polarization without Depolarization in High-Resolution MAS NMR[END_REF] However, the latter experiment was completed in ~ 16 hours, whereas the same experiment only took 20 minutes at 9.4 T (Figure 7). This clearly illustrates the need for further improvements in MAS-DNP efficiency, especially at high magnetic fields. NA ssNMR enabled by MAS-DNP is opening up exciting new avenues for the characterization of both biomolecular samples and materials. It has aided in structural measurements of protein fibrils, self-assembled small molecules, pharmaceuticals, heterogeneous catalysts, and more. With further innovation NA ssNMR will no doubt continue to be a useful tool in the structural biology and materials chemistry communities.

Figure 1 .

 1 Figure 1. Simulated effects of dipolar truncation on double quantum (DQ) filtered 13 C-13 C

Figure 2 .

 2 Figure 2. 2D DQ/SQ 13 C-13 C correlation spectra for a) uniformly 13 C, 15 N-enriched and b) NA

Figure 3 .

 3 Figure 3. Molecular formula of cyclic diphenylalanine a) and buildup of aromatic resonances b)

Figure 4 .

 4 Figure 4. At NA only spin-pairs need to be considered for numerical simulation of polarization buildup curves. a) 13 C spin system with different distances between nuclei in an isotopically

Figure 5 .

 5 Figure 5. DQ/SQ 13 C-13 C and 13 C-15 N correlation spectra of a) mutant huntingtin protein fibrils

Figure 6 .

 6 Figure 6. 1 H-13 C HETCOR spectrum of native collagen at NA. The highlighted cross peak (top left) denotes the correlation between 13 C from the rings of aromatic residues and 1 H's attached to

Figure 7 .

 7 Figure 7. DQ/SQ 13 C-13 C correlation experiments at NA of cellulose at a) 9.4 T and b) 18.8 T.

Table 1 .

 1 Natural isotopic abundance of commonly observed NMR active nuclei.

	Isotope Abundance (%) Nuclear spin (I)	γ (MHz/ T)	Receptivity relative to 1 H a
	1 H	99.985	1/2	42.58	1.0
	2 H	0.015	1	6.54	1.5 × 10 -6

The authors declare no competing financial interests.

ACKNOWLEDGMENT

This work was funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Action-795423-BOLD-NMR (to A.N.S.) and the European Research Council grant ERC-CoG-2015 No. 682895 (to G.D.P). In addition, this work was partially supported by funding from the French National Research Agency (ANR-11-LABX-0003-01, ANR-15-IDEX-02, and ANR-16-CE11-0030).

AUTHOR INFORMATION

Notes


Edith Archambault

Introduction

In France, like in other Western European countries, the third sector has been on a steady increase during the last decades. Similar to the situation in Germany, the French third sector is predominately financed by public money that is concentrated on those subsections which form the core of the welfare state: education, health and social services. However, public funding is currently being reduced and against this background, government and nonprofit organisations are looking for new ways of co-operation. In other words, in France the partnership of the third sector with government is changing as well as its position and role in the French welfare mix.

In the following, the results of the Johns Hopkins Nonprofit Sector Comparative Project for France will be presented, thus outlining:

the overall size of the third sector in France in 1995, -its composition, -its revenue sources, and its recent evolution.

Thereafter, the third sector's central role in labour market policies will be discussed. In France, many new jobs, new markets and new skills have initially been explored by volunteer work. Combined with a trend towards professionalisation, this knowledge served as a blueprint for paid labour. Moreover, the changing role of the nonprofit organisations as partners in a new welfare mix will be analysed. Centenary of 1901 Act gathered on July 1st 2 many nonprofit leaders, many government representatives and fourteen ministers including the Prime Minister to sign a Charter of reciprocal commitments between the government and the nonprofit sector. It was also a kind of celebration of the government/nonprofit sector partnership. But beyond this temporary event the relationship is not so clearly defined and in some subfields it includes a newcomer: the forprofit sector.

Dimension of the French nonprofit sector

As stated elsewhere (Archambault 1997a(Archambault , 1997b)), the French nonprofit sector developed only quite recently; associationsthe generic form for most nonprofit organisationsbeing illegal during the whole 19 th century until a centenarian act legalised them. During the 1960s and the 1970s the French Nonprofit Sector slowly grew in a context of state-provided welfare. More recently, the decentralisation of 1982 broke with a millenary Jacobean tradition which was a major incentive for the third sector to contract with local authorities. Graph 1 shows this recent growth: today between 60,000 and 70,000 associations are created per year, more than three times the average of the 1960s.

Graph 1: Annual creations of associations, 1960/2001

Source: Archambault, . 1 9 6 0 1 9 6 2 1 9 6 4 1 9 6 6 1 9 6 8 1 9 7 0 1 9 7 2 1 9 7 4 1 9 7 6 1 9 7 8 1 9 8 0 1 9 8 2 1 9 8 4 1 9 8 6 1 9 8 8 1 9 9 0 1 9 9 2 1 9 9 4 1 9 9 6 3

Overall size of the French third sector

In 1995 full-time-equivalent employment was 975,000 including religion and 960,000 excluding religionthat is 5 per cent of the total employment or the total employment of all consumption goods manufacturing industries in 1995.

In addition to this paid employment, eleven millions of volunteers are working in the third sector. One French individual out of four declare to be a volunteer.

Graph 2: Nonprofit share of total employment by country, 1995

Source: Salamon et al. (1998).

The total operating expenditures of the third sector in 1995 was FF 290 billion (44.2 billion Euro), which is the equivalent of the turnover of the public utilities industry or the mechanical engineering industry. These figures do not include the estimated monetary value of volunteering which would raise the sum by some 70 per cent. Therefore, the economic activity of the French third sector -which is frequently a by-product of its social role -is significant and sizeable. In a comparative perspective France is a "middle weight". Graph 2 shows the nonprofit share of total employment by country. The French third sector ranks exactly at the average level among the 22 countries included in the statistical part of the project. Germany is in a similar position. However, in comparison to other Western European countries, the French and German third sectors are developed below the average -due to the fact that education, a significant area of nonprofit activity elsewhere is primarily provided by public entities in both countries.

0 , 0 % 2 , 0 % 4 , 0 % 6 , 0 % 8 , 0 % 1 0 , 0 % 1 2 , 0 % 1 4 , 0 % R o m a n i a M e x i

Composition of the French third sector

The structure of the French nonprofit sector in 1995 can be seen in table 1. The first column lists the organisations, or, more precisely, the percentage of establishments included in the SIRENE file, i.e. organisations which employ at least one salaried or which pay taxes. Out of a total number of nonprofit organisations, which is unknown (maybe 800,000), about 250,000 organisations are included in SIRENE file. The other columns show the percentage of the total operating expenditures, the full-time equivalent employment (FTE), and volunteering (see above).

There are five significant features of the French nonprofit sector:

-From an economic point of view, the sector is concentrated on the core welfare activitiesculture and recreation, education and research, health and social serviceswhich represent 88 per cent of the total operating expenditures and the employment of the third sector, 80 per cent of the organisations recorded in SIRENE file, and 75 per cent of volunteer work. -Just as the German Third Sector, the French sector is dominated by social services, and this primacy has recently even been increased with the deepening of the economic depression in 1993. Nonprofit organisations run 55 per cent of the overall number of residential care facilities, with a quasi-monopolistic position in the field of residential facilities for disabled persons. -France has a higher percentage of third sector involvement in education than Germany has: about one fourth of the total resources of the nonprofit sector reside in the field of education. Catholic primary and secondary schools are over-represented in the western part of France, where the Belgian, or Dutch pattern prevails. All over the rest of France, private schools frequently represent a second chance for children rejected by public schools. Culture, sports and recreation are also more developed in France.

This sub-sector has been growing rapidly ever since the government started its policy of decentralisation. Many small organisations -run mainly by volunteers -are mushrooming everywhere. Due to the fact that hospitals have been secularised, especially during the French Revolution, the percentage of third sector organisations active in the field of health is much lower in France than it is in Germany. -There is a contrast between those sub-sectors which are highly professional like education, health and social services -where volunteer work still exists but at the margin, and where the division of labour is the rule -and those sub-sectors where volunteer work is the main resource, like culture, sports and recreation (which total nearly half of volunteering), or environment, international activities and professional associations. -Philanthropic intermediaries and, more generally, all kinds of foundations -grantmaking, corporate or operating -are less numerous in France than in other industrial countries because they have been repressed for centuries. Before 1987, there was no law truly governing foundations and today there exist less than 500 independent foundations in France. This support structure is typical of Western European countries. Table 3 shows that the nonprofit sectors of all Western European countries included in the Johns Hopkins Comparative Nonprofit Sector Projectexcept for Spainare government-dominant in their funding structure. France ranks at a low-average position among these countries: the relative share of public funding is smaller than it is in Ireland, Belgium, Germany or the Netherlands, but higher than it is in Austria and the UK. The overall composition of the resources of the French third sector hides a wide range of funding patterns among the sub-sectors as can be seen in graph 4. Health, education and social services are the only sub-sectors that are predominately funded by public money, they are also 'closer to the welfare state'. Other sub-ectors show a greater variety of sources of support: philanthropic intermediaries are mainly financed by giving while commercial resources provide for culture and recreation, development and housing, environment and professional associations. For civic associations public funding and commercial resources are balanced and the same is true for international activities where public funding -coming mainly from the European Unionand private giving are balanced.

The evolution of the French third sector (1990-1995)

Between 1990 and 1995, nonprofit employment has shown a rapid growth of 20 per cent, from 803,000 to 960,000 FTE wage-earners. This result is noteworthy because during the same period total employment has declined by one per cent. This trend of nonprofit employment (1990-95) has several causes:

-During this period of time, male employment has declined while female employment has increased, this trend benefitting nonprofit employment that is 70 per cent female. -Moreover, employment has declined not only in agriculture and manufacturing but also in commercial and financial service activities. Only relational service activities, either market or non-market, have shown a growth in employment. Nonprofit organisations provide mainly relational services: out of 157,000 jobs created by the third sector from 1990 until 1995 one half has been created in the social services and one sixth in the culture and recreation subsector. -Finally, the third sector has played a pioneering role in the labour market policies as we will see hereafter.

During the 1990-95 period, volunteer work has grown faster than paid employment, with a 10 per cent average annual growth. Its highest growth rate has occurred in the social services sector. Against the background of the deepening of depression, volunteer work appears as a way to obtain results rapidly at the local level, while public policies appear to be inefficient There has been little change in the composition of the nonprofit sector within five years; as mentioned before, the preponderance of social services has even been increased. The structure of revenue sources shows a slight trend towards the privatisation of resources: a little less public funding, a little more earned income. Against the background of the results of the Johns Hopkins Comparative Nonprofit Sector Project, the following section will focus on how nonprofit organisations are actively forming a partnership with public entities, specifically in health or social services.

New forms of partnership between the public and the nonprofit sector

For the two last decades nonprofit organisations have been partners of a new welfare mix. Today they play also an increasing role in labour market policies. Of course, these two points are linked. Francejust as Germanyis generally characterised as a corporatist welfare state (Esping [START_REF] Esping-Andersen | The Three Worlds of Welfare Capitalism[END_REF]: in order to acquire social rights, an individual has to have worked previously during a minimum duration. Therefore, in such a welfare system long-term unemployment means poverty -at the same token the access to the labour-market represents the key for social rights.

The position of the nonprofit organisations in labour market policies

During the last two decades, nonprofit organisations have secured their position -within or outside the framework of public labour market policies -in order to help specific categories of unemployed people to enter or re-enter in the labour market. One of the strategies of public labour market policy from 1991 onwards until today has been to offer part-time collective-utility jobs to young unemployed people or to the long-term unemployed. The wage bill is paid mainly by the government and symbolically by the recipient organisationbe it a public agency, a local community or a nonprofit organisation. The job must not compete with any regular paid job; training and supervision are to be ensured by the recipient organisation and half of the worker's time is to be devoted to job search and continuing education. Under these specific conditions nonprofit organisations have created many jobs, but evaluation studies show that, after the helped contracts, many of the participants of these programmes fall back into unemployment.

In 1997 these collective-utility jobs have become reserved for severe social cases and in 1998, more regular jobsalso paid mainly by the government -were offered to qualified young people (emplois-jeunes). Once more, nonprofit organisations acted as employers for these emplois-jeunes, After three years of existence, nonprofit organisations were the main employers of these jobs, after public schools and gymnasiums and before the police force.

The nonprofit organisations have acquired a position as job providers also outside the framework of public labour market policies. From the early 1980s onwards some nonprofit organisations have specialised in "integration through an economic activity, called entreprises d'insertion (integration enterprises) or associations intermédiaires (intermediary associations). The entreprises d'insertion provide goods or services in activities either overlooked by standard businesses or in competition with them; the associations intermédiaires supply temporary manpower to non-market employers, households, nonprofit organisations or public agencies. However, for both organisations, the main objective is to help the most severely deprived people to join the mainstream and maybe to enter the standard labour market through training programs and on-the-job training. These direct agents of employment policies deal with very difficult economic and social situations: unskilled school drop-outs, potential or former delinquents, alcoholics and drugaddicts. The main resource of these nonprofit organisations is the product of their economic activity but the time spent to train or follow-up the apprentices is compensated by tax or social contribution cuts and by grants. Evaluation studies show that the "integration through an economic activity" organisations have been more efficient than any other training programme in the 1980s, but their scope was reduced by the deepening of the depression during the early 1990s [START_REF] Archambault | Intermediaries Associations and Integration Enterprises[END_REF]. In 1998, a Law to fight deep poverty, prepared by the government with 18 specialised nonprofits, decided that the number of integration enterprises has to be doubled and proposed increased incentives Beyond this role in the professional and social integration of frail populations, nonprofit organisations are also creating new job opportunities through their standard activity -whatever its field.

For example, quite frequently nonprofit organisations are founded -by young people eager to be self-employed or by former executives who have some capital of their own -for the purpose of testing a business project. The very simple legal status of associations is used as an incubator for enterprises: if the project proves to be viable, the "forprofit in disguise" achieves a legal business status, it becomes a corporation or limited liability enterprise. In the same direction some associative or cooperative incubators where created to employ young people.

But even more importantly, nonprofit organisations have been, are and will be pioneers in exploring new service activities. These activities may become either market or public services or stay in the nonprofit sector. In this context a prime example is the Club Méditerranée that was at first a nonprofit organisation of social tourism. Moreover, many professional football clubs were initially nonprofit organisations. New skills are also frequently tested by volunteer work that turns into paid employment when the activity is growing, very often through public funding. This has been the case for social work during the interwar and postwar period and, more recently, for environmental jobs, sports and cultural management, training, or homework specialised in the aid of dependent people. The professionalisation-cycle is marked by the creation of professional associations, by new diplomas testifying the new skills, and sometimes by a specific collective bargaining. Let us note that two third of the 22 new jobs created by the emplois-jeunes have first been tested in nonprofit organisations. In that sense, volunteer work is a matrix or blue print of future labour, a testing area for flexible employment and, therefore, a significant element of transitional labour markets [START_REF] Schmid | Transitional Labour Markets. Concepts and Examples in Europe[END_REF]Gazier, 1998, Schmid and[START_REF] Schmid | The Dynamics of Full Employment; Social Integration through Transitional Labour Markets[END_REF].

Nonprofit organisations are partners in a new welfare mix

The development of a mixed provision system in the field of social services can be explained partly by a policy towards decentralisation which overcomes a thousand-year-old tradition of etatism, and partly by the initiatives of the nonprofit sector facing new social issues. On the one hand, the Decentralisation Acts (1982)(1983) have redistributed the responsibilities between the state and local governments. New activities and new resources were transferred to local governments 1 who on their part contracted out the provision of many services. The nonprofit sector has been the main partner in the field of culture and arts, sports, education and training, and, above all, social welfare services outside the social security system. This has to be seen against the background of the welfare state being severely criticised -in France as elsewherefor the inefficiency of public policies, the weight of bureaucracy, the tendency to create irresponsible beneficiaries and their inadequacy to cope with evolving needs.

According to Ullman (1993), the "delegation to nonprofit organisations has been the result of the deliberate effort on the part of political elites to improve the functioning and extend the scope of both social service provision and of French democracy (...) it was an effort to right the imbalance between an overly centralised and insular state and a weak and fragmented civil society". Also, public authorities have been overwhelmed by a growing demand for more diversified social services and again it has been the nonprofit organisations who were able provide specific types of services to more consumerist users. Moreover, the nonprofit sector has been very innovative in coping with new social issues. Since the social security scheme only deals with a limited number of social risks, it has been the third sector to first meet the needs of the handicapped, the rehabilitated alcoholics or drug-addicts, the pre-delinquent, the dependent elderly and so on.

More generally speaking, the third sector has coped with social prevention, social exclusion, and rural desertification at the local level because nonprofit organisations can mix social services delivery with advocacy far better than public agencies could do it. Nonprofits are also able to build new solidarity networks, for instance among the persons struck down by a rare disease or among the victims of the same natural or manmade disaster. Finally, they have access to specific resources, such as giving and volunteering, and to proximity saving networks or joint-ventures which again lowers their costs.

What kind of partnership results from this new welfare-mix? A labour division between the public and the nonprofit sector emerges according to which the state provides standard and basic services towards the whole population and also those services which are either highly medicalised or linked to regalian functions -law and public order or justice, for example. The third sector provides personal and specialised services directed to minority groups and endangered populations or services involving moral support, the amelioration of family relations or a labour-using following-up. According to the subsidiary principle -a newcomer in France -the bulk of resources of the nonprofit organisations providing these social services is coming from government at its different levels, with variations according to the subfields: public funding is higher for facilities and services intended for the disabled, while users fees cover a lager part for day care, elderly homes, or home care services.

The large-scale public funding in the field of social services is balanced by various regulations related to the creation, costs and activities of nonprofit establishments. Habilitation, accreditation, and agrément are different forms of contracts granted by the administration with a discretionary decision. They imply a control of the skills of the staff and of the quality of the delivered services. The reciprocal commitments are written down in those contracts. Nonprofit leaders regret that annual contracts can be revised by the administration unilaterally and ask for pluri-annual contracts which permit long-term planning. They also fear to become mere tools in the hands of public authorities. Conversely, central or local government ask for more accountability on the part of the nonprofit organisations.

While forprofit providers have firmly established the health field for several decades now, they still play only a small role in the provision of social services in France. The one exception are residential facilities for the elderly and home services where forprofits have been attracted by a more solvent population thanks to tax incentives. However, the competition between nonprofit and forprofit providers cannot be compared to the situation in UK or Italy, and tenders are not as common as in those countries. In France the trend towards "quasi-markets" was condemned by the left government (1997)(1998)(1999)(2000)(2001)(2002) which criticised tax incentives for home services as favouring the wealthy strata of population only. Accordingly, the government reduced the tax incentives so that private companies are no longer as interested in "proximity services" as they were some years ago.

Certainly, the most interesting role of the nonprofit organisations in the welfare partnership is their recent involvement in the definition of public policies as it has been the case with the 1975 Law on Disabled People, the 1988 Act creating a Minimum Income, and the 1998 Law Against Social Exclusion. Nonprofit organisations are considered to be the representatives of the underprivileged stratum of the population. Besides the labour-unionswhich are the representatives of the workers -they are the advocates of the out-of-work just as they are the advocates of the "-less": the homeless, the identity-paper-less, the right-less, the stateless...

Conclusion

To conclude on the new welfare mix, nonprofit organisations are trying to meet some of the new challenges of the XXI century. Amongst those are:

-First and foremost, the ageing of the population -by 2020 the number of dependent elderly is expected to be 1.2 million, nearly twice today's number. Community-care and continuing care will grow; they offer a trust-relationship and a co-ordination with the family that give a relative advantage to nonprofit organisations. Following the German position, the French social security has recently, in 2000, decided to cover the dependency risk by a specific benefit according to the level of dependency and means-tested. However the funding of dependency expenditures is still a mix of various resources. Recently, some nonprofit organisations have also specialised in the tutelage of dependent people. -The family policy is more and more oriented towards the conciliation of professional and family lives, and the nonprofit organisations have afforded many innovative responses to this problem. Their responses characteristically provide educational and recreational activities to the children and the youth during the out-of-school time. Nonprofit day-cares are also more flexible than the public ones are and more fitted to the various time-schedule of working parents -The social exclusion of unskilled young people and the urban deprived areas are also major social issues -we have seen above the role of nonprofit organisations in the social integration policy. Furthermore, the activities of immigrant nonprofit organisations aiming at equal opportunities for nationals and non-nationals in the fields of employment, housing, education and social life are crucial for the fight against racist or cryptoracist behaviours. -The growth of solitude in our society can be seen when comparing the successive population censuses. It is partly a consequence of the ageing of the population, partly of the growth of divorce and partly of the growing mobility of the population. This rising degree of solitude goes with less family solidarity and also less social rights, therefore it predisposes to poverty. By providing other neighbourhood social ties, nonprofit organisations can play a great part in solving this new social issue. -Finally, the third sector can help to build a social and citizen Europe as it is a pioneer on the way towards citizenship and everyday-democracy. Many nonprofit organisations are already involved in European networks, such as the Red Cross, the Caritas, or youth organisations. A European association legal status is on the agenda and European umbrellas appear step by step. Without any doubt the third sector has an important role to play in promoting citizenship and responsibility among its members and is able to balance self-interest,, ,the basis of a market-based economic union, by a sense of public interest, shared with the future political institutions.

Table 1 : Structure of the French nonprofit sector, 1995

 1

		Number of	Operating	FTE	Volunteering
	Sub-sector/Group	(SIRENE file) organisations	tures Expendi-	% of total employment	% of total
		% of total	% of total
	Culture and recreation	41.6 %	15.5 %	12.1 %	46.7 %
	Education and research	15.7 %	24.8 %	20.7 %	8.9 %
	Health	2.4 %	14.5 %	15.5 %	3.4 %
	Social services	20.7 %	32.8 %	39.7 %	15.7 %
	Total (the first 4 sub-sectors)	80.4 %	87.6 %	88.0 %	74.7 %
	Environment	3.2 %	1.0 %	1.0 %	8.7 %
	Development and hous-ing	3.8 %	4.4 %	5.5 %	4.0 %
	Civic and advocacy associations	6.1 %	2.6 %	1.9 %	1.8 %
	Philanthropic intermediar-ies	0.0 %	0.3 %	0.0 %	1.1 %
	International activities	0.4 %	1.3 %	1.8 %	3.0 %
	Professional associa-tions, unions	6.1 %	2.9 %	1.8 %	6.6 %
	Total (the last 6 sub-sectors)	19.6 %	12.4 %	12.0 %	25.3 %
	TOTAL	100 %	100 %	100 %	100 %

Revenue sources of the French third sector

Table 2

 2

and graph 3 show the overall importance of the three major sources of income of the French nonprofit sector: public funding, private earned income and private giving. With 58 per cent of the total, public resources have a large preponderance, coming primarily from social security, secondly from central government and, last but not least, from local governments. Private earned income covers slightly more than one third of the total resources: commercial resources -i.e. fees, charges and salesare the main component, followed by membership dues and finally, at a very low level, by investment income. Private giving is low: it covers only 7.5 per cent of the total resources of the French nonprofit sector, one half coming from individual contributions, the other half coming from corporate giving or sponsorship.

Table 2 and Graph 3: Sources of revenue of the nonprofit sector in France, 1995

 2

	Source	Amount (FF million)	%
	Public	166,000	58%
	Private	121,000	42%
	-earned	99,000	34,5%
	-giving	22,000	7,5%
	TOTAL	287,000	100%

Source: Johns Hopkins Nonprofit Sector Comparative Project -Phase 2

Table 3 : Sources of nonprofit revenue by country 1995 (19-country-average)

 3

		Private earned	Public sector	Private giving
	Fee-dominant
	Mexico	85%	9%	6%
	Peru	68%	19%	13%
	Australia	62%	31%	6%
	Japan	62%	34%	3%
	Finland	58%	36%	6%
	US	57%	31%	13%
	Slovakia	56%	21%	23%
	Hungary	55%	27%	18%
	Romania	54%	11%	36%
	Spain	49%	32%	19%
	Government-dominant
	Ireland	15%	78%	7%
	Belgium	18%	77%	5%
	Germany	32%	64%	3%
	Israel	26%	64%	10%
	Netherlands	36%	60%	2%
	France	35%	58%	7%
	Austria	44%	50%	6%
	UK	45%	47%	9%
	Czech Republic	40%	43%	18%
	TOTAL	47%	42%	11%

Source:

Salamon and al. Johns Hopkins Nonprofit Sector Comparative Project -1998

Graph 4: Revenue sources by subsector, 1995

	Health
	Education and research
	Social services
	Civic and advocacy associations
	International activities
	Development and housing
	Environment
	Culture and recreation
	Professional associations, unions
	Philanthropic intermediaries
	0%	10%	20%	30%	40%	50%	60%	70%	80%	90%	100%
			Public		Private earned	Private giving

Source: Johns Hopkins Nonprofit Sector Comparative Project -Phase 2.