This work is funded by the ANR (13-PDOC-0017-01 to B.H. and ANR-14-CE09-0020-01 to A.L.), &#34;Investments for the future&#34; Programme IdEx Bordeaux/CNRS (PEPS 2016 to B.H.) reference ANR-10-IDEX-03-02 to B.H., the Fondation pour la Recherche M&#233;dicale (FRM-AJE20140630090 to A.L.), the FP7 program (FP7-PEOPLE-2013-CIG to A.L.) and the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (ERC Starting Grant to A.L., agreement No 639020) and project &#34;WEAKINTERACT.&#34;

The authors have nothing to disclose.

Solid-state NMR (SSNMR) is a method of choice for characterizing macromolecular protein assemblies at an atomic level. One of the central issues in SSNMR-based structure determination is the spectral quality of the investigated system, that allows establishing 3D structural models of various precision, typically ranging from low-resolution models (containing the secondary structure elements and little 3D information) to pseudo-atomic 3D structures. The quantity and quality of structural information extracted from multi-dimensional SSNMR experiments is the key to compute a high-resolution NMR structure of the assembly.
The described protocol relies on the detection of 13C-13C and 15N-13C structural restraints requiring the recording of several 2D (and sometimes 3D) spectra with high signal-to-noise. At moderate MAS frequencies (&#60;25 kHz), the sample is introduced into rotors with sizes of 3.2-4 mm diameter allowing for protein quantities of up to ~50 mg, dependent on the sample hydration. The amount of sample inside the rotor is directly proportional to the signal-to-noise ratio in SSNMR spectra, a decisive factor for the detection of long-range distance restraints and their unambiguous assignment.
The spectral resolution is a crucial parameter during the sequential resonance assignment and the restraints collection. To obtain optimal results, the sample preparation parameters need to be optimized, particularly in the purification of the subunit and the assembly conditions (pH, buffer, shaking, temperature, etc.). For sample optimization, it is recommended to prepare unlabeled samples for several distinct conditions for which assembly has been observed, and to record a 1D 1H-13C CP spectrum (described in step 2.1) on each prepared sample. The spectra serve to compare spectral resolution and dispersion between the different preparations, based upon which the optimal conditions can be determined.
The quality of the SSNMR data depends strongly on the choice of the NMR acquisition parameters, especially for the polarization transfer steps. The use of high magnetic field strengths (&#8805;600 MHz 1H frequency) is essential for high sensitivity and spectral resolution, required when facing complex targets such as macromolecular protein assemblies.
A limiting factor in many cases is the spectrometer availability. Therefore, a judicious choice of the samples to be prepared should precede the spectrometer session. In any case, a uniformly 13C, 15N-labeled sample is a prerequisite to perform the sequential and intra-residual resonance assignment. For proteins assigned by solid-state NMR techniques see71. Structure determination of macromolecular assemblies at moderate MAS frequencies requires selectively 13C-labeled samples; for the detection of long-range 13C-13C and 13C-15N contacts samples based on 1,3-13C- and 2-13C-gylcerol and/or 1-13C - and 2-13C-glucose labeling are commonly used, as described above. The choice between the two labeling schemes is based on the spectral signal-to-noise ratio and resolution. To distinguish between intra- and intermolecular long-range contacts, mixed labeled and diluted samples have revealed efficient.
In short, the critical steps for an atomic SSNMR structural study are: (i) the preparation of the subunits and the assembly need to be optimized to obtain excellent sample quantity and quality, (ii) spectrometer field strength and acquisition parameters have to be chosen carefully; (iii) selective labeling strategies are required for a 3D structure determination and the amount of required data depends on data quality and the availability of complementary data.
Despite its applicability to a wide range of supramolecular systems ranging from membrane proteins to homomultimeric nano-objects, SSNMR is often limited by the need for mg-quantities of isotopically labeled material. The recent technological developments in ultra-fast MAS (&#8805;100 kHz) SSNMR open up the avenue to 1H-detected NMR, and push the limit of minimal sample quantity to sub-mg 72,73,74. Nevertheless, for detailed structural studies 13C-labeled samples are indispensable, which limits the application of SSNMR to samples assembled in vitro or to systems expressed in organisms that survive on minimal medium where in-cell SSNMR is an emerging method (for reviews see 75,76,77,78).
An important factor in SSNMR application to obtain high-resolution 3D structures is the spectral resolution: intrinsic conformational heterogeneity in an assembly can limit spectral resolution and spectra analysis. Residue specific 13C labeling may in some cases provide an alternative to obtain specific distance information on strategic residues in order to obtain structural models (for a recent examples see 79,80).
SSNMR for 3D structure determination still requires the collection of several datasets with often long data collection times on sophisticated instruments, depending on the approach and the system several days to weeks on a 600-1000 MHz (1H frequency) spectrometer. Therefore, the access to spectrometer time can be a limiting factor in an in-depth SSNMR study.
In the case of homomultimeric protein assemblies, leading to SSNMR data of sufficient quality to identify a high number of structural restraints such as in 3,57,64,70, SSNMR still gives no access to the microscopic dimensions. Therefore, in a de novo SSNMR structure determination of a homomultimeric assembly, EM or mass-per-length (MPL) data ideally complement SSNMR data to derive the symmetry parameters. SSNMR data alone provide the atomic intra- and intermolecular interfaces
SSNMR is highly complementary with structural techniques such as EM or MPL measurements but the data can also perfectly be combined with atomic structures obtained by X-ray crystallography or solution NMR on mutated or truncated subunits. An increasing number of studies can be found in literature where the conjunction of different structural data has allowed for determining atomic 3D models of macromolecular assemblies (see Figure 6 for representative examples).
In the field of Structural Biology, SSNMR emerges as promising technique to study insoluble and non-crystalline assemblies at the atomic level, i.e. providing structural data at the atomic scale. In this respect, SSNMR is the pendant to solution NMR and X-ray crystallography for molecular assemblies, including membrane proteins in their native environment and protein assemblies such as viral envelopes, bacterial filaments or amyloids, as well as RNA and RNA-protein complexes (see for example81). Its highly versatile applications in vitro and in the cellular context, such as tracking secondary, tertiary and quaternary structural changes, identifying interaction surfaces with partner molecules on the atomic scale (for example 82) and mapping molecular dynamics in the context of assembled complexes, indicate the important potential of SSNMR in future structural studies on complex biomolecular assemblies.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>M9 medium</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>0.5 g/L</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>3 g/L</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>6.7 g/L</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>10 &#956;M</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>1 &#956;M</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>MEM vitamin mix 100X</td>
			<td>10 mL/L</td>
		</tr>
		<tr>
			<td>13C-glucose</td>
			<td>2 g/L</td>
		</tr>
		<tr>
			<td>15NH4Cl</td>
			<td>1 g/L</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of minimal expression medium for recombinant protein production in E. coli BL21 cells.

Advances in magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (SSNMR) offer an efficient tool for the structural characterization of macromolecular protein assemblies at an atomic resolution. These protein assemblies are ubiquitous systems that play essential roles in many biological processes. Their molecular structures, interactions and dynamics are accessible by SSNMR studies, as has been shown for viral (capsids1) and bacterial infection mechanisms (secretion systems2,3, pili4), membrane protein complexes5,6,7,8 and functional amyloids 9,10,11. This type of molecular assembly can also provoke pathologies such as in neurodegenerative diseases where proteins assemble in misfolded, amyloid states and cause aberrant cell behavior or cell death 12,13. Protein assemblies are often built by the symmetric oligomerization of multiples copies of protein subunits into large supramolecular objects of various shapes including fibrils, filaments, pores, tubes, or nanoparticles. The quaternary architecture is defined by weak interactions between protein subunits to organize the spatial and temporal assembly and to allow for sophisticated biological functions. Structural investigations at an atomic scale on these assemblies are a challenge for high-resolution techniques since their intrinsic insolubility and very often their non-crystallinity restricts the use of conventional X-ray crystallography or solution NMR approaches. Magic-angle spinning (MAS) SSNMR is an emerging technique to obtain atomic resolution data on insoluble macromolecular assemblies and has proven its efficiency to resolve 3D atomic models for an increasing number of complex biomolecular systems including bacterial filaments, amyloid assemblies and viral particles 14,15,16,17,18,19,20,21,22. Technical advances on high magnetic fields, methodological developments and sample preparation has established MAS SSNMR into a robust method to investigate insoluble proteins in various environments, notably in their biologically-relevant macromolecular assembled state or in cellular membranes, making the technique highly complementary to cryo-electron microscopy. In many cases, a very high degree of symmetry characterizes such protein assemblies. MAS SSNMR exploits this feature, as all protein subunits in a homomolecular assembly would have the same local structure and therefore virtually the same SSNMR signature, drastically reducing the complexity of the analysis.
An efficient protocol for structural studies of macromolecular protein assemblies by moderate MAS (&#60;25 kHz) SSNMR is presented in this video and can be subdivided into different steps (Figure 1). We will demonstrate the critical stages of the workflow of a SSNMR structural study exemplified on a filamentous protein assembly (see highlighted steps in Figure 1), with the exception of protein subunit purification, differing for each protein assembly but of critical importance for structural studies, and without going into the technical/methodological details of SSNMR spectroscopy and structure calculation for what specialized tutorials are available online. While the present protocol will primarily focus on solid-state NMR experiments performed under MAS conditions, the use of aligned biological environments 23,24,25,26,27, such as aligned bicelles, allow for the investigation of protein conformation and dynamic protein-protein interaction in membrane-like media without MAS technology. We will show the protein expression and assembly steps as well as the recording of the crucial SSNMR spectra and their analysis and interpretation. Our aim is to provide insights into the structural analysis pipeline enabling the reader to perform an atomic-resolution structural study of a macromolecular assembly by SSNMR techniques.
The protocol encompasses 3 sections:
1. Solid-state NMR sample production
As a prerequisite to a solid-state NMR analysis, the protein components of the macromolecular assembly need to be expressed, isotope-labeled, purified and assembled in vitro into the native-like complex state (for an example see Figure 2). To ensure high NMR sensitivity, isotope enrichment in 13C and 15N labeling is required through the use of minimal bacterial expression media supplemented with 13C and 15N sources, such as uniformly 13C-labeled glucose/glycerol and 15NH4Cl respectively. In the later stage of the protocol, selectively 13C-labeled samples produced with selectively 13C-labeled sources such as (1,3-13C)- and (2-13C)-glycerol (or (1-13C)- and (2-13C)-glucose) are used to facilitate the NMR analysis. Mixed labeled sample corresponding to an equimolar mixture of either 50% 15N- and 50% 13C-labeled or 50% (1,3-13C)- and 50% (2-13C)-glucose are introduced to describe the detection of intermolecular interactions. A high degree of protein purity as well as rigorous conditions during the assembly step are key factors to insure a homogeneous structural order of the final sample.
2. Preliminary structural characterization based on one-dimensional (1D) solid-state NMR
We present the essential experiments for a structural analysis by SSNMR. One-dimensional (1D) cross-polarization (CP) and INEPT / RINEPT28 experiments, detected on 13C nuclei are used to detect rigid and flexible protein segments in the assembly, respectively, and to estimate the degree of structural homogeneity and local polymorphism (for an example see Figure 3).
3. Conformational analysis and 3D structure determination
Subsections 1 and 2 concern the conformational analysis, which is based on the SSNMR resonance assignment of all rigid residues of the protein assembly, as the chemical shifts are very sensitive probes to the local environment and can be used to predict the phi/psi dihedral angles and thereby determine the secondary structure. Figure 4 illustrates an example of a sequential resonance assignment in the rigid core of a protein assembly. The 3D structure determination is based on the collection of structural data such as distance restraints encoding close proximities (&#60;7 - 9 &#197;), containing both intra- and intermolecular information. Subsections 3 and 4 describe long-range distant restraint collection and interpretation. Long-range contacts are defined as intramolecular 13C-13C proximities arising from residue i to j, with |i-j| &#8805;4, defining thereby the tertiary protein fold of the monomeric subunit, or as intermolecular 13C-13C proximities, defining the intermolecular interfaces between protein subunits in the assembly. Intra- and intermolecular interfaces are illustrated in Figure 5. SSNMR restraints detected through 13C-13C and 15N-13C recoupling experiments usually encode for internuclear distances &#60; 1 nm. Subsection 4 explains the detection of intermolecular distance restraints. In symmetrical protein assemblies, the use of homogeneously labeled samples (i.e. 100% uniformly or selectively labeled) for identifying intermolecular subunit-subunit interactions is limited, as both intra- and inter-molecular contacts lead to detectable signals. The unambiguous detection of intermolecular proximities is achieved by using mixed labeled samples, containing an equimolar mixture of two differently labeled samples, combined prior to aggregation. Subsection 5 briefly introduces structure modeling.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55779/55779fig1.jpg" /> 
Figure 1:&#160;Workflow of an atomic-resolution structural study by solid-state NMR.13C,&#160;15N isotope labeled protein production, subunit purification, subunit assembly, control of assembly formation, SSNMR experiments, SSNMR experiment analysis and extraction of distance restraints, and structure modeling are shown.&#160;<a href="//cloudflare.jove.com/files/ftp_upload/55779/55779fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="741">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Instruments</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">NMR Spectrometer (&#62; 11.7 Tesla)</td>
			<td style="width:117px;">Bruker</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">triple resonance MAS SSNMR probehead&#160;</td>
			<td style="width:117px;">Bruker</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">SSNMR rotors 4mm</td>
			<td style="width:117px;">Bruker</td>
			<td style="width:119px;">K1910</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Centrifuge 5804 R&#160;</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:119px;">5805000629</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">GeneQuant 1300 spectrometer</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">28-9182-13</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">IGS60 INCUBATEUR HERATHERM 75 L</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">228001</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MaxQ 4450 bench top orbital shaker</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">78376</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tube Revolver Agitator</td>
			<td style="width:117px;">Dutscher</td>
			<td style="width:119px;">79547</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">sonopuls HD 3100</td>
			<td style="width:117px;">Bandelin</td>
			<td style="width:119px;">3680</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MicroPulser electroporator</td>
			<td style="width:117px;">Biorad</td>
			<td style="width:119px;">165-2100&#160;</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">mini-PROTEAN tetra cell system</td>
			<td style="width:117px;">Biorad</td>
			<td style="width:119px;">165-8000</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AKTA pure system</td>
			<td style="width:117px;">GE Healthcare</td>
			<td style="width:119px;">29-0182-24</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">capillary microman M25 pipet</td>
			<td style="width:117px;">Gilson&#160;</td>
			<td style="width:119px;">F148502</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Materials</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:251px;">amiconR ultra-15</td>
			<td style="width:117px;">sigma</td>
			<td style="width:119px;">Z740199-8EA</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">capillaries and pistons</td>
			<td style="width:117px;">Gilson&#160;</td>
			<td style="width:119px;">F148112</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">spatula</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">13263799</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Reagents</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:251px;">D-glucose 13C6</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">389374</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ammonium-15N-chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">299251</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:251px;">1,3 13C2 glycerol</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">492639</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:251px;">2 13C glycerol</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">489484</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Kanamycin</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">K1876</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Carbenicillin</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">C3416</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium phosphate dibasic</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">S7907</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium phosphate monobasic</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">71380</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">calcium chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">C1016</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Magnesium sulfate</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">208094</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iron Chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">157740</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Zinc chloride</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">793523</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MEM Vitamin Solution (100&#215;)</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">M68954</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">IPTG</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">BP1755</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Trizma base&#160;</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricine</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">T0377</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">SDS</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">436143</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">sodium azide</td>
			<td style="width:117px;">sigma</td>
			<td style="width:119px;">71289</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">4,4-dimethyl-4-silapentane-1-sulfonic acid&#160;</td>
			<td style="width:117px;">Sigma</td>
			<td style="width:119px;">178837</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Name</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Softwares</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Unicorn 6.3</td>
			<td style="width:117px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">Akta systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">ccpNMR</td>
			<td style="width:117px;">CCPN&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">spectrometer systems</td>
		</tr>
	</tbody>
</table>

atomic scale structural studies of macromolecular assemblies by solid-state nuclear magnetic resonance spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

The typical SSNMR workflow includes several steps illustrated in Figure 1. Usually the protein subunits are produced by in vitro heterologous expression in E. coli, purified and assembled under shaking but sometimes also in static conditions. Expression and purification of the protein subunit are followed by SDS gel chromatography (Figure 2A). The formation of macromolecular assemblies can then be confirmed by electron microscopy (EM) analysis (see Figure 2B for an example of a filamentous assembly).
After introduction of the protein assembly into the SSNMR rotor, the rotor is inserted into the spectrometer, the MAS frequency and temperature are regulated and the spectra are recorded. First insights can be obtained by 1D SSNMR techniques. Figure 3 shows a typical SSNMR FID detected on the 13C channel on a structurally homogeneous protein sample, a 1H-13C CP spectrum, revealing the13C resonances present in the rigid core of the protein subunit in the assembly, and a 2D 1H-13C INEPT spectrum, representing the mobile residues. For atomic insights into the rigid core of the assembly structure, multidimensional SSNMR experiments need to be recorded on uniformly and selectively labeled samples to first assign the SSNMR resonances and then detect long-range proximities (see Figure 4).
All spectra are processed and analyzed with adequate software to assign the SSNMR resonances and extract intra- and intermolecular distance restraints (Figure 5). The SSNMR distance restraints are either used alone or in conjunction with data from complementary techniques, which can be integrated into the modeling program.
For representative atomic structures of macromolecular assemblies solved by SSNMR techniques Figure 6 illustrates several filamentous assemblies from bacterial appendages and amyloid fibrils.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55779/55779fig6v2.jpg" /> 
Figure 6:Filamentous macromolecular structures determined by a solid-state NMR approach: bacterial filaments and amyloid protein fibrils.A) Type 1 pilus of uropathogenic E. coli, PDB code 2N7H 4; B) ASC filament, PDB code 2N1F 63; C) Type III secretion system needles, PDB codes 2MME, 2LPZ and 2MEX 2,3,64; D) Amyloid-beta AB42 fibrils, PDB code 2NAO, 5KK3, 2MXU 65,66,67 and Osaka mutant PDB code 2MVX 57, Iowa mutant PDB code 2MPZ 58; E) Alpha-synuclein fibrils, PDB code 2N0A 68; F) HET-s prion domain, PDB code 2RNM, 2KJ3 69,70. <a href="https://cloudflare.jove.com/files/ftp_upload/55779/55779fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

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15.3K Views.  UMR5248 CNRS, Université de Bordeaux. Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Video: Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...