Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

Summary

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.

Abstract

NEMO is a scaffolding protein which plays an essential role in the NF-κB pathway by assembling the IKK-complex with the kinases IKKα and IKKβ. Upon activation, the IKK complex phosphorylates the IκB molecules leading to NF-κB nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-κB pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

Introduction

The NF-κB pathway is activated in response to a variety of stimuli, including cytokines, microbial products and stress, to regulate expression of target genes responsible for inflammatory and immune response, cell death or survival and proliferation¹. Pathologies including inflammatory and autoimmune diseases and cancer^2,3,4,5 have been correlated to hyperactivation of the pathway, which has made modulation of NF-κB activity a prime target for the development of new therapies^6,7.

The canonical NF-κB pathway in particular is distinguished from the non-canonical pathway, responsible for lymphorganogenesis and B-cell activation, by the former's dependence on the scaffolding protein NEMO (NF-κB essential modulator⁸) for the assembly of the IKK-complex with the kinases IKKα and IKKβ. The IKK complex is responsible for the phosphorylation of IκBα (inhibitor of κB) that targets it for degradation, freeing the NF-κB dimers to translocate to the nucleus for gene transcription¹ and is therefore an attractive target for the development of inhibitors to modulate NF-κB activity.

Our research focuses on the characterization of the protein-protein interaction between NEMO and IKKβ, targeting NEMO for the development of small molecules inhibitors of IKK complex formation. The minimal binding domain of NEMO, required to bind IKKβ, encompasses residues 44-111, and its structure has been determined in complex with a peptide corresponding to IKKβ sequence 701-745⁹. NEMO and IKKβ form a four-helix bundle where the NEMO dimer accommodates the two helices of IKKβ(701-745) in an elongated open groove with an extended interaction interface. IKKβ(734-742), also known as the NEMO-binding domain (NBD), defines the most important hot-spot for binding, where the two essential tryptophans (739,741) bury deeply within the NEMO pocket. The details of the complex structure can aid in the structure-based design and optimization of small molecule inhibitors targeting NEMO. At the same time, it is difficult that binding of a small molecule or peptide would recreate in NEMO the full conformational change (i.e., extensive opening of the NEMO coiled-coil dimer) caused by binding of the long IKKβ(701-745), as observed in the crystal, and the structure of unbound NEMO or NEMO bound to a small molecule inhibitor may represent a better target for structure-based drug design and inhibitor optimization.

Full length NEMO and smaller truncation constructs encompassing the IKK-binding domain have proven intractable for structure determination in the unbound form via X-ray crystallography and nuclear magnetic resonance (NMR) methods¹⁰, which prompted us to design an improved version of the IKK-binding domain of NEMO. Indeed, NEMO (44-111) in the unbound form is only partially folded and undergoes conformational exchange and we therefore set to stabilize its dimeric structure, coiled-coil fold and stability, while preserving binding affinity for IKKβ. By appending three heptads of ideal dimeric coiled-coil sequences¹¹ at the N-and C-termini of the protein, and a series of four point mutations, we generated NEMO-EEAA, a construct fully dimeric and folded in a coiled coil, which rescued IKK-binding affinity to the nanomolar range as observed for full length NEMO¹². As an additional advantage, we hoped the coiled-coil adaptors (based on the GCN4 sequence) would facilitate crystallization and eventually aid in the X-ray structure determination via molecular replacement. Coiled-coil adaptors have been similarly utilized to both increase stability, improve solution behavior and facilitate crystallization for trimeric coiled coils and antibody fragments^13,14. NEMO-EEAA is easily expressed and purified from Escherichia. coli cells with a cleavable Histidine tag, is soluble, folded in a stable dimeric coiled coil and is easily crystallized, with diffraction to 1.9 Å. The presence of the ordered coiled-coil regions of GCN4 could additionally aid in phasing the data from crystals of NEMO-EEAA by molecular replacement using the known structure of GCN4¹⁵.

Given the results obtained with apo-NEMO-EEAA, we believe the protocols described here could also be applied to the crystallization of NEMO-EEAA in the presence of small peptides (like the NBD peptide) or small molecule inhibitors, with the goal of understanding the requirements for NEMO inhibition and structure-based optimization of initial lead inhibitors to high affinity. Given the plasticity and dynamic nature of many coiled-coil domains¹⁶, the use of the coiled-coil adaptors could find more general applicability in aiding structural determination.

Protocol

1. Design of construct for crystallography

1. Clone the sequence of NEMO-EEAA as in previous publication¹² in a vector for expression in E. coli using the T7 promoter, including a N-terminal hexa-histidine tag and a protease cleavage site.
   NOTE: In this protocol, we used a vector modified to include a N-terminal hexa-histidine tag and a Tobacco Etch Virus (TEV) cleavage site¹⁰. This vector facilitates cleavage of the His tag for protein crystallization and leaves only the short extension of GSW residues before the start of the desired protein sequence. The vector from which this was derived, and alternative vectors are listed in the Table of Materials. In this protocol, subsequent modifications to the original NEMO(44-111) sequence were introduced stepwise, as described earlier¹⁰, using side directed mutagenesis. We initially attempted to stabilize the NEMO coiled-coil dimer appending the ideal coiled-coil adaptors (in a length of at least three heptads) to the N-terminal or C-terminal end or to both. The double coiled coil was the most promising from earlier crystallization trials and it was subsequently modified introducing mutations to improve crystallization as described previously¹².

2. Large scale expression of His₆ tagged NEMO-EEAA

1. Transform construct into BL21(DE3) competent cells. Store at -80 °C as a cell glycerol stock.
2. Day 1 – Prepare a cell starter culture. In a 125 mL Erlenmeyer flask, add 20 mL of Terrific Broth solution and 20 µL of a 100 mg/mL stock of Ampicillin. Add a few microliters of cell glycerol stock (from -80 °C storage of BL21(DE3) competent cells transformed with vector).
3. Shake the 10 mL starter culture overnight at 37 °C, 220 rpm (approximately 15 h).
4. Day 2 – From the starter, dilute to an OD₆₀₀ = 0.1 in 250 mL of Terrific Broth. Add ampicillin to a final concentration of 100 µg/mL. Grow to an OD₆₀₀ = 0.8-1.0.
   1. Add isopropyl β-D-1-thiogalactopyranoside (IPTG) to 500 µM, and grow for 4 h at 37 °C.
   2. Measure OD₆₀₀ of induced culture after 4 h. Culture should reach an OD₆₀₀ = 6-10.

3. Purification of His₆ tagged NEMO-EEAA

1. Spin cell culture down at 3,800 x g for 20 min at 4 °C.
2. Save the cell pellet and discard the medium.
   NOTE: The cell pellet can be saved and stored at -20 °C at this point, for purification at a later time.
3. Resuspend the cells in 40 mL of lysis buffer containing 20 mM Tris, 150 mM NaCl, 10 mM imidazole, 2 mM MgCl₂, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM Dithiothreitol (DTT), and 3 µL of Benzonase Nuclease.
   NOTE: The Nickel immobilized metal ion affinity chromatography (IMAC) column utilized is compatible with 2 mM DTT. Alternatively, 0.2 mM tris(2-carboxyethyl)phosphine (TCEP) can be utilized.
4. Split resuspended cells into two 20-25 mL aliquots.
5. Lyse the cells using French press (approximate pressure 25,000 psi), repeating 2-3 times for each aliquot (in the cold room).
   NOTE: Alternatively, cells could be lysed by sonication (not tested in this protocol).
6. Add urea to the cell lysate to a final concentration of 8 M, allow to incubate on a rocking platform for a minimum of 2 h or up to overnight. This and all following purification steps, with the exception of the dialysis, can be performed at room temperature.
7. Day 3 – Transfer the lysate to ultracentrifuge tubes and balance weight, ensuring lysate fills tubes to at least ¾ full. Spin the lysate at 125,000 x g for 45 min at 25 °C. Decant the supernatant into a 100 mL beaker for loading onto column.
   NOTE: Centrifuging at 4 °C will cause urea to crash out.
8. On a fast liquid chromatography system, remove ethanol from IMAC 5 mL column with 25 mL of ultrapure H₂O at 5 mL/min, followed by 25 mL of elution buffer containing 20 mM Tris, 150 mM NaCl, 500 mM imidazole, 2 mM DTT, pH 8.0, then 25 mL of binding buffer containing 20 mM Tris, 150 mM NaCl, 10 mM imidazole, 2 mM DTT, 8 M urea, pH 8.0.
9. Load urea incubated supernatant at 3 mL/min onto IMAC column, collecting the flow through. Wash the column for 10 column volumes with binding buffer at 3 mL/min.
10. Refold NEMO-EEAA construct on column by washing column with refolding buffer for 20 column volumes at 3 mL/min, containing 20 mM Tris, 150 mM NaCl, 10 mM imidazole, 2 mM DTT, pH 8.0.
11. Perform gradient elution of NEMO-EEAA, from 10 to 500 mM Imidazole over a 12-column volume gradient, collecting all eluate in fraction collection plate (1 mL fractions).
12. Continue elution at 500 mM imidazole for two column volumes, continuing to collect.
13. Run sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) of fractions to determine NEMO- EEAA presence in elution fractions.
    NOTE: We used 10% acrylamide, MES buffer.
14. Pool fractions containing pure target protein.
15. Measure protein concentration by Bradford assay¹⁷.
    NOTE: This is necessary to estimate the amount of protease for tag cleavage.

4. His₆ tag cleavage and purification

1. Add TEV in a 1:10 weight ratio of TEV:NEMO-EEAA protein to cleave the His₆ tag. The TEV protease was purified in house.
   NOTE: Optimize the amount of TEV, time and temperature required for complete cleavage separately.
   1. Express TEV protease, S219V mutant¹⁸ in BL21(DE3)-RIL cells and purify as described earlier¹⁹. Briefly grow the cells as described in step 2, lyse by French press and purify using an IMAC column. Store final protein in 25 mM sodium phosphate buffer, pH 7.8, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 20% v/v glycerol.
2. Dialyze the sample overnight (approximately 15 h) in 4 L of 20 mM Tris, 150 mM NaCl, 2 mM DTT, pH 8.0, to allow for cleavage and to remove excess imidazole from the sample for the subsequent purification.
3. Day 4 – Remove the sample from dialysis. Run an SDS-PAGE gel of the sample from TEV cleavage to ensure cleavage is completed.
4. On a fast liquid chromatography system, remove ethanol from an IMAC 5 mL column with 25 mL of ultrapure H₂O at 5 mL/min, followed by 25 mL of elution buffer containing 20 mM Tris, 150 mM NaCl, 500 mM imidazole, 2 mM DTT, pH 8.0, then binding buffer containing 20 mM Tris, 150 mM NaCl, 10 mM imidazole, 2 mM DTT, pH 8.0.
5. Load the column at 1 mL/min with TEV-cleaved NEMO-EEAA. Cleaved NEMO-EEAA will elute in the flow-through: collect in a 96 well fraction collection plate (1 mL fractions). Wash the column for five column volumes of 20 mM Tris, 150 mM NaCl, 10 mM imidazole at 1 mL/min, continuing to collect in fraction collection plate.
6. Elute TEV and uncleaved His₆-NEMO-EEAA with three column volumes of 20 mM Tris, 150 mM NaCl, 500 mM imidazole, 2 mM DTT, pH 8.0, collecting elution in a 50 mL flask.
7. Run SDS-PAGE gel of flow-through fractions to determine presence of cleaved NEMO-EEAA.
8. Pool flow-through fractions containing cleaved NEMO-EEAA construct, and concentrate using a stirred-cell concentrator to 5 mL. Membrane molecular weight cut-off (MWCO) = 3 kDa.
9. Using a 3 kDa MWCO membrane, dialyze concentrated sample in 2 L of 20 mM Tris, 100 mM NaCl, 2 mM DTT, pH 8.0 for 2 h. Change the dialysis buffer to 2 L of fresh dialysis buffer for overnight dialysis (approximately 15 h), at 4 °C.
10. Day 5 – Load 5 mL of the dialyzed sample on a size exclusion chromatography (SEC) 16 mm x 60 cm column (34 μm average particle size) at 1 mL/min in 2 mM Tris, 100 mM NaCl, 2 mM DTT, pH 8.0. Repeat with additional columns depending on sample volume.
11. Pool the fractions corresponding to dimeric NEMO-EEAA.
    NOTE: NEMO-EEAA elutes between 60-65 mL, corresponding to a larger molecular weight protein, due to the elongated nature of the dimeric coiled coil.
12. Concentrate using a stirred-cell concentrator and a MWCO = 3 kDa membrane to a final concentration of 113 µM (1.65 mg/mL).
13. Aliquot the protein and store at 4 °C (stable for over 1 month).

5. Sparse matrix screening

NOTE: The protocol performs crystallization trials using commercially available screens and setting up sitting drop experiments using a crystallization robot. Crystal images are collected automatically by an imager.

1. Using commercially available sparse matrix screens (see Table of Materials), pipette 60 µL of sparse matrix solution into each of the 96 wells of a 2 drop-chamber crystallization plate for sitting drop vapor diffusion (reservoir solution).
2. Using a robotic drop setter, dispense 100 nL of protein solution at 1.65 mg/mL in a 1:1 ratio with reservoir solution in drop 1 for a final volume of 200 nL; then 66 nL of protein solution with 134 nL of reservoir solution for a final volume of 200 nL in drop 2 (1:2 ratio).
3. Seal the plate using 3-inch-wide sealing tape immediately after dispensing.
   NOTE: Drops will dry out if left exposed to atmosphere for longer than 2-3 min.
4. Store the trays in the crystallization imager storage, at 20 °C, checking the images collected automatically for crystal presence, starting after two days.
   NOTE: Crystallization screening proceeded in parallel with construct optimization. Initial crystals formed in the following conditions (commercial screens listed in the Table of Materials): a) 0.1 M Tris, pH 8.0, 30 % v/v polyethylene glycol (PEG) MME 550, 5% poly-γ-glutamic acid, 200-400 kDa low molecular weight polymer (PGA -LM); b) 0.1 M Tris, pH 7.8, 20% w/v PEG MME 2k, 5% PGA-LM; c) 0.1 M Tris, pH 7.8, 20% w/v PEG 3350, 5% PGA-LM. Sparse matrix screen crystals will have poor lattice uniformity; therefore, they will look poor using cross-polarized imaging. Use UV imaging to ensure that the crystals contain protein. The following seed stock generation step is necessary for obtaining the final diffraction quality crystals.

6. Seed stock generation

NOTE: We reproducibly obtain crystals for seed generation in 0.1 M Tris pH 8.0, 5% PGA-LM, 3.6% w/v PEG 20k. However, crystals will show high mosaicity and are unsuitable for data collection at this stage.

1. Using a kit for seed generation, prepare seed stock by pipetting out entire drop with crystal present, and place into 50 µL of crystallization condition solution in the provided vial.
2. Vortex the seed stock for 3 min, pulsing 20 s on and 10 s off.
3. Serially dilute seed stock in 1:10 increments down to 1:10,000. Store all dilutions at 4 °C, for further use.

7. Fine screens

1. Design fine screens varying the conditions for Tris, PGA-LM, and PEG. Vary PEG length for individual trays.
   NOTE: The fine screen that produced the crystal utilized for structure determination of NEMO-EEAA employed the following conditions: 0.1 M Tris pH 8; PGA-LM varied from 8 to 0% in columns 1-12. Rows A-H screened different PEGs, with concentrations varying in columns 1-12 as follows. A: PEG 200 (0-40% v/v); B: PEG 400 (0-40% v/v); C: PEG MME 500 (0-40% v/v); D: PEG 1000 (0-30% w/v); E: PEG 3350 (0-30% w/v); F: PEG 6k (0-30% w/v); G: PEG 10k (0-20% w/v); H: PEG 20k (0-20% w/v). The crystal appeared in well H4 (5.45% w/v PEG 20k, 5.8% w/v PGA-LM). In this protocol, a protein crystallography screen builder liquid handler was used to build the screens.
2. Seed a protein stock in a 1:25 volume ratio of 1:1,000 seed dilution.
   NOTE: Concentration of NEMO-EEAA will drop slightly, but crystals will still form.
3. Repeat step 2 using 1:10,000 seed dilution, same 1:25 volume ratio.
4. Using the drop setter, dispense 100 nL of protein solution at 1.65 mg/mL, with 1:1,000 dilution of seed stock into a 1:1 ratio with reservoir solution in drop 1 for a final volume of 200 nL. Repeat for drop 2, but with 1:10,000 dilution of seed stock present.
5. Seal the plate using 3-inch-wide sealing tape immediately after dispensing.
   NOTE: Drops will dry out if left exposed to atmosphere for longer than 2-3 min.
6. Store the trays in the imager storage, at 20 °C, checking images collected after two days for crystal presence.
7. Check the crystals with the cross-polarizer imager for lattice uniformity, to select for single conditions to set up following trays.

8. Generation of crystals for data collection

1. Design single condition screens around Tris, PGA-LM, and PEG condition which produced uniform crystals as analyzed by cross-polarized images, and the largest crystals possible.
   NOTE: Crystals from these conditions are irregular in shape, mostly rectangular thin sheets. It is key to select a condition where the edges of the crystal are well defined and have the greatest thickness possible.
2. Make 20 mL of crystallization condition by hand.
3. Using a multi-channel pipettor, dispense 60 µL per well in a 2 drop-chamber, 96 well crystallization plate for sitting drop vapor diffusion.
4. Prepare seed stock as described in step 6.2.
5. Dispense the protein as described in step 6.3.
6. Seal the plate using 3-inch-wide sealing tape immediately after dispensing.
   NOTE: Drops will dry out if left exposed to atmosphere for longer than 2-3 min.
7. Store the trays in the imager at 20 °C and check the images for crystal presence every day.
8. Check cross-polarized images of the crystals for lattice uniformity, to select crystals for data collection.

9. Determination of cryo-protectant

1. To test the cryo-protectants, create stock solutions corresponding to the crystallization conditions but containing 30%, 20%, 10%, and 5% higher concentration of each component. The addition of the cryo-protectant volume will result in a final concentration of components that is the same as the crystallization conditions.
   NOTE: For testing a cryo-protectant at a 30% concentration by volume for a 0.1 M Tris crystallization condition, start with a stock solution of 0.143 M Tris, before adding the cryo-protectant.
2. From a cryo-reagents kit, create 10 µL of sample for cryo-protectant test by mixing 30% by volume of cryo condition in 70% of crystallization condition stock solution, for a final concentration of 30% cryo-protectant in original crystallization conditions. Mix thoroughly.
   1. Using a 10 µL pipette, take 5 µL of test cryo-protected solution and plunge the pipet tip into liquid nitrogen. If ice is observed, discard.
   2. Test all cryo-protectants at 30%. For the successful solutions, repeat the process at 20%, then 10% and 5% of cryo-protectant.
   3. Utilizing the successful cryo-protectant solution with the smallest percentage of cryo-protectant, add 0.5 µL of solution into a test drop with crystals present. Observe under microscope, timing how long crystal lasts in the condition, if not indefinite.
      NOTE: These crystals are for test only and will be discarded. For NEMO-EEAA, 12% 1,2-propanediol is the optimal cryo-protectant solution. 12% accounts for the dilution the cryo-protectant solution will experience when added to the crystal drop of approximately 100 nL, for an approximate final concentration of 1,2-propanediol of 10%.

10. Crystal looping

1. Loop crystals 1-2 days before shipment to synchrotron.
   NOTE: Crystals will be roughly 60-100 µm in diameter: 0.05 – 0.10 mm loops are ideal for looping.
2. Cut the tape from the top of the well.
3. Add 0.5 µL of crystallization solution containing 12% 1,2-propanediol cryo-protectant directly to the well.
   NOTE: Final concentration of 1,2-propanediol is now at approximately 10%, due to dilution by 100 nL of drop solution (approximate drop volume reduction from initial 200 nL, due to vapor diffusion).
4. Loop the crystal from the well.
   NOTE: Crystals often grow on the bottom of the well but will dislodge with a gentle nudge from the loop. Once dislodged, loop.
5. Store the crystal containing cryo-loops in pucks immersed in liquid N₂.
6. Store these pucks in liquid N₂ in Dewar flask until they are ready for shipment for X-ray diffraction at the synchrotron.

11. Data collection

1. Collect X-ray diffraction data. In this protocol, use AMX beamline (ID: 17-ID-1), National Synchrotron Light Source II.
   NOTE: Data was collected on site but can be collected remotely. Collect data in the region of the crystal which showed lattice uniformity in cross-polarized images. Position the crystals in the loop so that data collection does not involve "poor" regions of the crystal while the crystal rotates in the goniometer. Data which provided the best resolution came from collection on the edge of an extension of a crystal about 5 x 5 μm in size. Use rastering to identify the best areas on the crystal to collect²⁰.

12. X-ray data processing

1. Process dataset to highest resolution collected (1.8 Å) with XDS IDXREF program to determine space group, unit cell, and solvent content.
2. Integrate data using XDS INTEGRATE program.
3. Process scaled intensities from XDS XSCALE program using STARANISO Server, using I/σI cutoff mean of 1.2 for diffraction-limit surface for the data.
   NOTE: Data will not be cut in true ellipsoid. Retain all data above the I/σI of 1.2 cutoff. The statistics on the data calculated for spherical completeness will be poor, due to the non-spherical truncation. The elliptical completeness was 88% with highest resolutions of: 1.88 Å, 2.10 Å and 2.55 Å along the a*, b* and c axis, respectively.

13. Structure solution

1. Utilize the X-ray structure of GCN4 (PDB: 4DMD)¹⁵ as a search model for molecular replacement using MRage²¹ in PHENIX²². The 4DMD structure was defined in MRage as an "ensemble", and the MRage solution successfully built the structure portion corresponding to the N-terminal coiled-coil adaptor of NEMO-EEAA, homologous to the search model, for both chains in the dimer.
   NOTE: Turn off anisotropy correction in PHASER, or data will be further scaled back.
2. Utilize successive rounds of Autobuild²³ in PHENIX and manual building (using 2F_o-F_c and F_o-F_c maps, in Coot²⁴) to build the remainder of the structure.
3. Manually build the residues still missing into the model based on 2F_o-F_c and F_o-F_c maps, using Coot²⁴.
   NOTE: The last stage involved building the 4 N-terminal residues and 4 C-terminal residues for each monomer. Sidechain placement was also manually adjusted as needed.
4. Calculate a composite omit map using PHENIX and a 10% omission of the structure.

14. Structure refinement

1. Refine the structures with PHENIX Refine. Run the initial refinements against bulk-solvent and stereochemistry weights, relaxing RMSbond and RMSangle constraints to 0.01 and 1.0, respectively. Continue refinement on Individual B-factors, TLS parameters, and Occupancies.

Results

Cloning, expression and purification of the IKK-binding domain of NEMO.
The protocol followed in this study to obtain the final sequence of NEMO-EEAA (Figure 1A), which produced diffraction quality crystals, involved the expression and characterization of all the intermediate constructs, including the addition of the coiled-coil adaptors at N- and or C-terminus, the mutations C76A, C95S and the mutations E56A, E57A. Figure 1

Discussion

Crystallization attempts of NEMO in the unbound form were unsuccessful, including attempts using the full-length protein and several truncation constructs encompassing the IKK-binding domain. Our biophysical characterization of the IKK-binding domain of NEMO (residues 44-111) by circular dichroism, NMR spectroscopy and fluorescence anisotropy indicated that the construct, albeit able to bind IKKβ, existed in a state of conformational exchange, not suitable for crystallization^9,

Materials

Name	Company	Catalog Number	Comments
20% w/v γ-PGA (Na+ form, LM)	Molecular Dimensions	MD2-100-150	For fine screen crystallization of NEMO-EEAA
3.5 kDa MWCO Dialysis Membrane	Spectra/Por	132724	For dialysis removal of imidazole
Amicon Stirred Cell	Millipose Sigma	UFSC 05001	For protein concentration
Ammonium Chloride	Millipore Sigma	G8270	For minimal media labeling
Benzonase Nuclease	Millipore Sigma	9025-65-4	For digestion of nucleic acid
BL21-CodonPlus (DE3)-RIL Competent Cells	Agilent Technologies	Model: 230245	TEV expression
CryoPro	Hampton Research	HR2-073	Cryo-protectants kit
D-Glucose (Dextrose)	Millipore Sigma	A9434	For minimal media labeling
Difco Terrific Broth	ThermoFisher	DF043817	For culture growth
Dithiothreitol > 99%	Goldbio	DTT25	For reduction of disulfides
E. coli: Rosetta 2 (DE3)	Novagen	71400-3	Expression of unlabeled NEMO-EEAA
FORMULATOR	Formulatrix		Liquid handler/ screen builder
HCl – 1.0 M Solution	Hampton Research	HR2-581	For fine screen crystallization of NEMO-EEAA
HiLoad 16/600 Superdex 75 pg	GE Healthcare	28989333	For size exclusion purification
HisTrap HP 5 mL column	GE Healthcare	17524802	For purification of His-tagged NEMO-EEAA
HT 96 MIDAS	Molecular Dimensions	MD1-59	For sparse matrix screening of NEMO-EEAA
HT 96 Morpheous	Molecular Dimensions	MD1-46	For sparse matrix screening of NEMO-EEAA
Imidazole	ThermoFisher	288-32-4	For elution from His-trap column
Isopropyl-beta-D-thiogalactoside	Goldbio	I2481C5	For induction of cultures
MRC2 crystallization plate	Hampton Research	HR3-083	Crystallization plate
NT8 - Drop Setter	Formulatrix		Crystallization
pET-16b	Millipore Sigma	69662	For cloning of NEMO-EEAA
pET-45b	Millipore Sigma	71327	For cloning of NEMO-EEAA
Phenylmethylsulfonyl fluoride	ThermoFisher	36978	For inhibition of proteases
Polycarbonate Bottle for use in Ultracentrifuge Rotor Type 45 Ti	Beckmann Coulter	339160	Ultracentrifuge bottle
Polyethylene Glycol 20,000	Hampton Research	HR2-609	For fine screen crystallization of NEMO-EEAA
pRK793 (TEV)	Addgene	Plasmid 8827	For TEV production
QuikChange XL II	Agilent Technologies	200522	Site directed mutagenesis
Required Cap Assembly:	Beckmann Coulter	355623	Ultracenttrifuge bottle cap
ROCK IMAGER	Formulatrix		Crystallization Imager
Seed Bead Kit	Hampton Research	HR2-320	Seed generation
Sodium Chloride ≥ 99%	Millipore Sigma	S9888	For buffering of purification solutions
TCEP (Tris (2-Carboxyethyl) phosphine Hydrochloride)	Goldbio	TCEP1	Reducing agent
The Berkeley Screen	Rigaku	MD15-Berekely	For sparse matrix screening of NEMO-EEAA
The PGA Screen	Molecular Dimensions	MD1-50	For fine screen crystallization of NEMO-EEAA
Tris – 1.0 M Solution	Hampton Research	HR2-589	For fine screen crystallization of NEMO-EEAA
Ultrapure Tris Buffer (powder format)	Thermofisher	15504020	For buffering of purification solutions
Urea	ThermoFisher	29700	For denaturation of NEMO-EEAA