This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.
Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.
Ribosomes are large ribonucleoprotein machines that carry out the essential role of translating mRNA into proteins in all living cells. Ribosomes are composed of two subunits which are produced in a complex process termed ribosome biogenesis1,2,3,4. Eukaryotic ribosome assembly relies on the aid of hundreds of essential ribosomal assembly factors2,3,5. Nsa1 (Nop7 associated 1) is a eukaryotic ribosome assembly factor that is specifically required for the production of the large ribosomal subunit6, and is known as WD-repeat containing 74 (WDR74) in higher organisms7. WDR74 has been shown to be required for blastocyst formation in mice8and the WDR74 promoter is frequently mutated in cancer cells9. However, the function and precise mechanisms of Nsa1/WDR74 in ribosome assembly are still largely unknown. To begin to uncover the role of Nsa1/WDR74 during eukaryotic ribosome maturation, multiple structural analyses were performed, including X-ray crystallography and small angle X-ray scattering (SAXS)10.
X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, electron microscopy, and SAXS are all important techniques for studying macromolecular structure. Size, shape, availability, and stability of macromolecules influences the structural biology method for which a particular macromolecule will be best suited, however combining multiple techniques through a so-called "hybrid" approach is becoming an increasingly beneficial tool11. In particular X-ray crystallography and SAXS are powerful and complementary methods for structural determination of macromolecules12.
Crystallography provides high-resolution atomic structures ranging from small molecules to large cellular machinery such as the ribosome, and has led to numerous breakthroughs in the understanding of the biological functions of proteins and other macromolecules13. Furthermore, structure-based drug design harnesses the power of crystal structures for molecular docking by computational methods, adding a critical dimension to drug discovery and development14. Despite its broad applicability, flexible and disordered systems are challenging to assess by crystallography since crystal packing can be hindered or electron density maps may be incomplete or of poor quality. Conversely, SAXS is a solution-based and low-resolution structural approach capable of describing flexible systems ranging from disordered loops and termini to intrinsically disordered proteins12,15,16. Considering it is compatible with a broad range of particle sizes12, SAXS can work synergistically with crystallography to expand the range of biological questions that can be addressed by structural studies.
Nsa1 is suitable for a hybrid structural approach because it contains a well-structured WD40 domain followed by a functional, but flexible C-terminus which is not amenable to X-ray crystallography methods. Following is a protocol for the cloning, expression, and purification of S. cerevisiae Nsa1 for hybrid structural determination by X-ray crystallography and SAXS. This protocol can be adapted to study the structures of other proteins that are comprised of a combination of ordered and disordered regions.
1. Recombinant Protein Production and Purification of Nsa 1
2. Crystallization and Proteolytic Screening of Nsa 1
3. X-ray Diffraction Data Collection and Nsa 1 Structure Solution
4. SAXS Data Collection, Processing, and Modeling
Nsa1 was PCR amplified from S. cerevisiae genomic DNA and subcloned into a vector containing an N-terminal 6x-Histidine affinity tag followed by MBP and a TEV protease site. Nsa1 was transformed into E. coli BL21(DE3) cells and high yields of protein expression were obtained following induction with IPTG and growth at 25 °C overnight (Figure 1A). Nsa1 was affinity-purified on immobilized cobalt affinity resin, followed by MBP cleavage with TEV protease, and finally resolved by size exclusion chromatography (Figure 1B). Fractions from size exclusion chromatography containing Nsa1 were pooled, concentrated to 8 mg/mL and then used for crystallization trials with a crystallization robot. Initial sparse matrix crystal screens yielded two different crystal forms of Nsa1, cubic and orthorhombic (Figure 2A).
During the optimization of the cubic and orthorhombic crystals, it was discovered that the orthorhombic crystals arose as the result of proteolytic cleavage of Nsa1. Limited proteolysis and mass spectrometry were used to determine the region of Nsa1 that was sensitive to proteolysis, and it was observed that Nsa1 was sensitive to a concentration gradient of the protease elastase (Figure 2B). Subsequent mass spectrometry analysis confirmed that this degradation resulted from loss of the C-terminus of Nsa1. A series of C-terminal truncations of Nsa1 were generated, to remove the proteolytic sensitive C-terminus (Figure 2C). The orthorhombic crystals could be repeated with the Nsa1ΔC (residues 1-434) truncation, which was ultimately used for SAD structure determination. The orthorhombic crystals could also be repeated by treating Nsa1FL with elastase for 1 hour at 4 °C prior to setting up crystal trays.
The cubic Nsa1 crystals were optimized using Nsa1FL through a combination of sodium citrate gradients, coupled with microseeding (Figure 3A). This yielded large, reproducible cubic crystals, with a diffraction limit of around 2.8 Å resolution (Figure 3C, left). The orthorhombic crystals could only be optimized using the C-terminal truncation variants of Nsa1, by varying the concentration gradients of PEG 1500 and PEG 400, combined with microseeding, which yielded large crystals with a diffraction limit of around 1.25 Å resolution (Figure 3A-C). Experimental phases of Nsa1 were determined by SeMet-SAD from a SeMet-derivative of Nsa1ΔC10.
The N-terminal seven-bladed β-propeller WD40 domain of Nsa1 was well resolved in both the cubic and orthorhombic crystal structures, however both structures lacked electron density for the C-terminus of Nsa1. SAXS was then used to determine the position of the missing C-terminal domain of Nsa1 in solution. After optimization of sample concentration for data collection, the partial atomic structure was used to perform rigid-body modeling, and generate an ab initio reconstruction of the missing components. The model was evaluated in terms of the goodness of the fit for the calculated scattering curves to the experimental data (Figure 4, center pipeline). The WD40 domain of Nsa1 alone is not a good fit of the experimental SAXS data, as evidenced by the discrepancy between the experimental scattering curve with the theoretical scattering curve, which was generated from crystal structure PDB ID 5SUM (Figure 4, left pipeline). In addition to rigid-body modeling, ensemble modeling was also done. This produced an ensemble of 3 to 4 conformers of Nsa1 and resulted in a lower χ2 value (Figure 4, right pipeline). The reduction in model discrepancy (χ2) using ensemble modeling revealed the conformational sampling of the Nsa1 C-terminal tail in solution.
Figure 1. Expression and purification of Nsa1 with a 6X-His-MBP fusion tag. (A) SDS-PAGE analysis of the protein expression in BL21 (DE3) cells at 25 °C overnight and the first purification step using cobalt affinity resin. (B) Representative size exclusion chromatogram following TEV cleavage. The fractions from size exclusion chromatography were analyzed by SDS-PAGE. The fractions from peak 1 containing Nsa1 were collected and used for structural analysis. Please click here to view a larger version of this figure.
Figure 2. The C-terminus of Nsa1 is sensitive to proteolysis. (A) Initial crystallization trials of Nsa1 yielded two different crystals forms: cubic and orthorhombic. UV microscope was used to verify that the crystals contained protein. Vis: Visible light, UV: UV microscopes. Scale bar = 50 µm in each window. (B) Proteolytic screening analyzed by SDS-PAGE. Three dilutions (1:10, 1:100, 1:1000) for each protease stock (0.1, 0.01, 0.001 mg/mL) were combined with aliquots of protein (1 mg/mL) to be screened. Protease resistant domains were analyzed the by SDS-PAGE and mass spectrometry after 37 °C incubation for 60 min. Nsa1-FL: fresh purified protein, Nsa1Δ: purified protein stored at 4 °C for 3 weeks after which a degraded form of the protein was observed. (C) Schematic diagram of the Nsa1 full-length (upper) and the C-terminal truncation construct (lower). Please click here to view a larger version of this figure.
Figure 3. Nsa1 Crystallization Optimization. (A) A seed stock was prepared from initial small crystals and used to make a dilution series (1x ~ 1/104x) (microseeding). By mixing 1 µL of protein with 1 µL of the diluted seed stock, the bigger single crystals grew within a week. (B) Precipitant concentration gradient for orthorhombic crystal optimization. (C) Optimized cubic and orthorhombic crystals used for data collection. Green circles indicate the typical area of the crystal trays which yielded data collection quality crystals. Please click here to view a larger version of this figure.
Figure 4. Schematic of Nsa1 SAXS Analysis. Overview of the pipeline used to process SAXS data and generate models with BUNCH (center) and EOM (right). The left pipeline shows the discrepancy between the experimental scattering curve (red circles, protein concentration: 6 mg/mL) with the theoretical scattering curve (blue line), which was generated from the crystal structure PDB ID 5SUM. The models were evaluated by comparing the experimental SAXS scattering curve (red circles, protein concentration: 6 mg/mL) with the scattering curve derived from the BUNCH model of Nsa1 (black line) or the EOM conformers of Nsa1 (black line). In each model, the WD40 domain is shown in cartoon colored in green (PDB ID 5SUM), the flexible C-terminus is shown in spheres colored in red for BUNCH and green, magenta, cyan, and yellow for the individual EOM conformers. The fraction of each conformer derived from EOM is labeled next to the model. Please click here to view a larger version of this figure.
Using this protocol, recombinant Nsa1 from S. cerevisiae was generated for structural studies by both X-ray crystallography and SAXS. Nsa1 was well-behaved in solution and crystallized in multiple crystal forms. During the optimization of these crystals, it was discovered that the C-terminus of Nsa1 was sensitive to protease degradation. The high resolution, orthorhombic crystal form could only be duplicated with C-terminal truncation variants of Nsa1, likely because the flexible C-terminus of Nsa1 prevented crystal packing. The structure of Nsa1 was solved by X-ray crystallography to high resolution, but the C-terminus could not be built in either crystal form because it was not ordered. Crystallography is the premiere technique for determining atomic resolution structures of macromolecules around the size of Nsa1, however as with any method, crystallography does have some limitations. One of the major limitations of crystallography is the inability to resolve disordered regions of proteins40,41.
The C-terminus of Nsa1 is important for proper nucleolar localization of the protein, underscoring the need to study its structure10. The C-terminus of Nsa1, was resolved by SAXS, a complementary structural biology technique to X-ray crystallography. SAXS data was recorded for full-length Nsa1 across a concentration series. From this concentration series, the optimal concentration for Nsa1 SAXS data collection and processing was determined. SAXS data were recorded for Nsa1 at 6, 4.5, and 3.0 mg/mL. The Guinier region, P(r) function and molecular weight were determined across the concentration series to ensure that the sample was well-behaved and not aggregated under the experimental conditions tested. To reconstruct the full-length structure of Nsa1, the theoretical scattering amplitude was determined from the partial crystal structure and then ab initio methods were used to model the flexible C-terminus. From this hybrid approach, it was determined that the flexible C-terminus of Nsa1 extends outward from the ordered WD40 domain.
Advances in processing tools has driven the popularity of SAXS for macromolecular structural studies. SAXS measures the X-ray scattering pattern from randomly oriented protein in solution to provide low-resolution structural information, including molecular mass and overall shape. Consequently, SAXS has emerged as a powerful orthogonal structure validation tool for crystallography. This is largely due to the development of computational methods to calculate the theoretical scattering of atomic structures and comparing them to experimental SAXS data37. Using this approach, the conformational state, quaternary structure, and higher-order assembly observed in a crystal lattice can be compared to the structural characteristics of the particle in solution. Furthermore, disordered loops and termini missing in high-resolution structures determined by X-ray crystallography can be modeled using solution scattering data. This hybrid structural approach uses the crystal structure as a building block for SAXS-guided modeling of missing residues and has proven to be effective in mapping the C-terminus of Nsa110, as well as other macromolecules such as the influenza A virus M1 matrix protein42and DEAD-box RNA chaperones43. Advanced SAXS-based modeling software can also address more complex systems, such as intrinsically disordered proteins, by mapping the conformational landscape of these systems using a series of conformers that together contribute to the overall scattering potential of the particle in solution16,39. Taken together, recent advances in SAXS data collection and processing tools contributes to the success of hybrid structural biology approaches for tackling challenging biological systems.
The combination of solution scattering with high resolution structures is poised to answer important questions about the flexibility and dynamics of macromolecules44. Many proteins, such as Nsa1, have dynamic regions that are important for biological function. In this manuscript a template protocol is provided which details the combination of SAXS with high-resolution structure determination by X-ray crystallography. In addition to X-ray crystallography SAXS can also be used to compliment other structural biology techniques including NMR, electron paramagnetic resonance (EPR), and fluorescence resonance energy transfer (FRET), further highlighting the importance of SAXS as a complementary structural biology technique45,46,47.
The authors have nothing to disclose.
Diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID and 22-BM beamlines at the Advanced Photon Source (APS), Argonne National Laboratory. The SAXS data was collected on the SIBYLS beamline at the Advance Light Source (ALS), Lawrence Berkeley National Laboratory. We would like to thank the staff at the SIBYLS beamline for their help with remote data collection and processing. We are grateful to the National Institute of Environmental Health Sciences (NIEHS) Mass Spectrometry Research and Support Group for help determining the protein domain boundaries. This work was supported by the US National Institute of Health Intramural Research Program; US National Institute of Environmental Health Sciences (NIEHS) (ZIA ES103247 to R. E. S.) and the Canadian Institutes of Health Research (CIHR, 146626 to M.C.P). Use of the APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. W-31-109-Eng-38. Use of the Advanced Light Source (ALS) was supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Additional support for the SIBYLS SAXS beamline comes from the National Institute of Health project MINOS (R01GM105404) and a High-End Instrumentation Grant S10OD018483. We would also like to thank Andrea Moon and Dr. Sara Andres for their critical reading of this manuscript.
Molecular Cloning of Nsa1 | |||
pMBP2 parallel vector | Sheffield et al, Protein Expression and Purification 15, 34-39 (1999) | We used a modified version of pMBP2 which included an N-terminal His-tag (pHMBP) | |
S. cerevisiae genomic DNA | ATCC | 204508D-5 | |
Primers for cloning Nsa1 | |||
SC_Nsa1_FLFw | IDT | CGC CAA AGG CCT ATGAGGTTACTAGTCAGCTGTGT GGATAG |
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SC_Nsa1_FLRv | IDT | AATGCAGCGGCCGCTCAAATTTT GCTTTTCTTACTGGCTTTAGAAGC AGC |
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SC_Nsa1_DeltaCFw | IDT | GGGCGCCATGGGATCCATGAGG TTACTAGTCAGCTGTGTGG |
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SC_Nsa1_DeltaCRv | IDT | GATTCGAAAGCGGCCGCTTAAAC CTTCCTTTTTTGCTTCCC |
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Recombinant Protein Production and Purification of Nsa1 | |||
Escherichia coli BL21 (DE3) Star Cells | Invitrogen | C601003 | |
pMBP- NSA1 and various truncations | Lo et al., 2017 | ||
Selenomethionine | Molecular Dimensions | MD12-503B | |
IPTG, Dioxane-Free | Promega | V3953 | |
EDTA Free Protease Inhibitor Cocktail | Sigma-Aldrich | 4693159001 | |
Sodium Chloride | Caledon Laboratory Chemicals | 7560-1-80 | |
Magnesium Chloride hexahydrate | Sigma-Aldrich | M2670 | |
Tris Buffer, 1 M pH7.5 | KD Medical | RGF-3340 | |
Glycerol | Invitrogen | 15514-029 | |
beta-mercaptoethanol | Sigma | M6250 | |
1M Imidazole, pH 8.0 | Teknova | I6980-06 | |
Talon Affinity Resin | Clonetech | 635503 | |
Amicon Ultra 15 mL Centrifugal Filter (MWCO 10K) | Millipore | UFC901024 | |
HiLoad 16/600 Superdex 200 Prep Grade Gel Filtration Column | GE-Healthcare | 28989335 | |
TEV Protease | Prepared by NIEHS Protein Expression Core | Expression plasmid provided by NCI (Tropea et al. Methods Mol Biology, 2009) | |
4-15% Mini-PROTEAN TGX Precast Protein Gels | BioRad | 456-8056 | |
Crystallization, Proteolytic Screening | |||
Crystal Screen | Hampton Research | HR2-110 | |
Crystal Screen 2 | Hampton Research | HR2-112 | |
Salt Rx | Hampton Research | HR2-136 | |
Index Screen | Hampton Research | HR2-144 | |
PEG/Ion Screen | Hampton Research | HR2-139 | |
JCSG+ | Molecular Dimensions | MD1-37 | |
Wizard Precipitant Synergy | Molecular Dimensions | MD15-PS-T | |
Swissci 96-well 3-drop UVP sitting drop plates | TTP Labtech | 4150-05823 | |
3inch Wide Crystal Clear Sealing Tape | Hampton Research | HR4-506 | |
Proti-Ace Kit | Hampton Research | HR2-429 | |
PEG 1500 | Molecular Dimensions | MD2-100-6 | |
PEG 400 | Molecular Dimensions | MD2-100-3 | |
HEPES/sodium hydroxide pH 7.5 | Molecular Dimensions | MD2-011- | |
Sodium Citrate tribasic | Molecular Dimensions | MD2-100-127 | |
22 mm x 0.22 mm Siliconized Coverslides | Hampton Research | HR3-231 | |
24 Well Plates with sealant (VDX Plate with Sealant) | Hampton Research | HR3-172 | |
18 mM Mounted Nylon Loops (0.05 mm to 0.5 mM) | Hampton Research | HR4-945, HR4-947, HR4-970, HR4-971 | |
Seed Bead Kit | Hampton Research | HR2-320 | |
Magnetic Crystal Caps | Hampton Research | HR4-779 | |
Magnetic Cryo Wand | Hampton Research | HR4-729 | |
Cryogenic Foam Dewar | Hampton Research | HR4-673 | |
Crystal Puck System | MiTeGen | M-CP-111-021 | |
Full Skirt 96 well Clear Plate | VWR | 10011-228 | |
AxyMat Sealing Mat | VWR | 10011-130 | |
Equipment | |||
UVEX-m | JAN Scientific, Inc. | ||
Nanodrop Lite Spectrophotometer | Thermo-Fisher | ||
Mosquito Robot | TTP Labtech | ||
Software/Websites | |||
HKL2000 | Otwinoski and Minor, 1997 | ||
Phenix | Adams et al., 2010 | ||
Coot | Emsley et al., 2010 | ||
ATSAS | Petoukhov et al., 2012 | https://www.embl-hamburg.de/biosaxs/atsas-online/ | |
Scatter | Rambo and Tainer, 2013 | ||
Pymol | The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. | ||
BUNCH | Petoukhov and Svergun, 2005 | ||
CRYSOL | Svergun et al, 1995 | ||
PRIMUS | Konarev et al, 2003 | ||
EOM | Tria et al, 2015 |