Preparation of scFv stabilized chromatosomes for single-particle cryo-EM structure determination

Summary The chromatosome, a nucleosome bound to a histone H1, is the structural unit of metazoan chromatin. Determination of the high-resolution structure of the chromatosome is challenging due to the dynamic nature of H1 binding. Here, we present a protocol for purifying an optimized single-chain antibody variable fragment (scFv) that can be used to stabilize the chromatosome for single-particle cryo-EM studies. This protocol facilitates high-resolution cryo-EM structure determination of nucleosomes with a natural DNA sequence, chromatosomes, and other protein nucleosome complexes. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2021).


SUMMARY
The chromatosome, a nucleosome bound to a histone H1, is the structural unit of metazoan chromatin. Determination of the high-resolution structure of the chromatosome is challenging due to the dynamic nature of H1 binding. Here, we present a protocol for purifying an optimized single-chain antibody variable fragment (scFv) that can be used to stabilize the chromatosome for single-particle cryo-EM studies. This protocol facilitates high-resolution cryo-EM structure determination of nucleosomes with a natural DNA sequence, chromatosomes, and other protein nucleosome complexes. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2021).

BEFORE YOU BEGIN
We previously demonstrated that a single-chain variable fragment (scFv) of a nucleosome antibody (Zhou et al., 2019) can stabilize the nucleosome with a natural DNA sequence, which enabled us to determine the cryo-EM structure of a native-like nucleosome to a global resolution of 2.6 Å . However, the original scFv construct (scFv 15 ) ( Figure 1A) is not stable and gradually forms aggregations when stored at 4 C (note, scFv 15 cannot be stored in frozen form as it completely aggregates upon thawing). Here we optimized the construct by switching the order of Hv and Lv fragments or by changing the length of the flexible linker (Krebber et al., 1997) ( Figure 1B). Remarkably, we found that one of the constructs (scFv 20 ) is much more stable. In our hands, scFv 20 can be stored at 4 o C for nearly a year without forming any apparent aggregations. In this protocol, scFv represents this optimized scFv 20 , unless stated otherwise.

Timing: 5-7 days
For this protocol, one should prepare four core histones following the previously published protocol (Dyer et al., 2004). Below are the steps for purifying the 197 bp W601 DNA from a plasmid (pUC19-16x197_W601) and using it for nucleosome reconstitution.
Preparation of 197bp W601 DNA CRITICAL: A plasmid with an array of repeat sequences tends to be unstable when propagated in bacterial strains, therefore it is critical to utilize a stable cell line such as NEB Stable cells or Invitrogen One Shot stbl2 cells. After transformation, check the plasmid isolated from several colonies by restriction enzyme digestion or DNA sequencing. This analysis is equally critical for the production of the desired DNA with high yield.
1. Transform 1 mL of pUC19-197x16_W601 plasmid ($100 ng/mL) into 50 mL NEB Stable cells following the manufacturer's protocol and spread around $20 mL cells onto an LB agar plate containing 100 ug/mL ampicillin. Incubate the plate in a 37 C incubator for $18 h. 2. Label 4 colonies separately, then inoculate each colony into 10 mL of LB medium containing 100 ug/mL ampicillin in a 50 mL Falcon tube. Grow in a 37 C incubator shaker at 220 rpm for 6 -8 h. 3. Transfer 4 mL of cell culture from each tube to four new Falcon tubes. Extract plasmids using the Promega Mini-Prep Kit, following the manufacturer's protocol. 4. Digest 200 ng of each purified plasmid using PstI and EcoRI restriction enzymes, which should yield a 3.2 kb insert (16 3 197 bp) band and a 2.7 kb pUC vector band when resolved in a 2% agarose gel (Figure 2, left, lanes 1-4). Plasmid digested with SmaI restriction enzyme alone should produce a 197 bp W601 DNA band and 2.9 kb vector band ( Figure 2, right, lanes 1-4). 5. Once the plasmids are verified, the corresponding cell cultures can be combined and equally distributed to six flasks containing 1 L 2x YT cell culture medium. Incubate the cell cultures in a 37 C incubator shaker set at 250 rpm for $18-24 h. 6. Harvest the cells by centrifugation at 4000xg, 4 C for 30 min in an Eppendorf 5810R centrifuge.
Perform large scale plasmid preparation by following the published protocol (Dyer et al., 2004). 7. The typical yield of plasmid from 6 L of cell culture is $80 -120 mg. To release the 16 copies of 197 bp W601 DNA, digest the plasmid with 10 U SmaI restriction enzyme per mg plasmid in 1x NEB Cutsmart buffer, adjust the plasmid concentration to $2 mg/mL with deionized (DI) water, and incubate in a 37 C incubator for 18 h. 8. Analyze the SmaI plasmid digestion on a 2% agarose gel. The presence of multiple bands other than the 197 bp DNA and 2.9 kb plasmid DNA suggests that the digestion is incomplete. In this case, add 50% more enzyme and incubate another 18 h. 9. Once the digestion is complete, Aliquot to $20 mL in several 50 mL Falcon tubes. Precipitate the plasmid backbone (2.9 kb band) by stepwise adding 40% PEG 6000 to the digestion solution ranging from 4% to 6%, while simultaneously adding 4 M NaCl to a final concentration of 0.5 M. Mix well. When the mixture turns cloudy, place the tubes on ice for at least 1 h to further precipitate the plasmid backbone DNA. 10. Pellet the plasmid backbone DNA by centrifugation at 4000xg, 4 C for 30 min in an Eppendorf 5810R centrifuge. Decant $15 mL of supernatant to each 50 mL Falcon tube and precipitate the 197 bp DNA by adding $30 mL of cold ethanol. 11. Pellet the 197 bp DNA by centrifugation at 4000xg, 4 C for 30 min in an Eppendorf 5810R centrifuge, discard the supernatant, wash the pellet with 70% ethanol three times, air dry the pellet and then redissolve it in TE 10/1 (10 mM Tris, 1 mM EDTA, pH8.0) buffer. 12. Extract the DNA once with phenol:chloroform:isoamyl alchohol (25:24:1) (PCIA) and then with chloroform:isoamyl alcohol (CIA) to remove residual enzyme and PEG 6000. Precipitate the DNA using ethanol, and then redissolve it in TE 10/1 buffer. The typical yield of 197 bp DNA is $30 -40 mg. Store the 197 bp DNA at 4 o C at a concentration of $ 5 mg/mL.
Alternatives: DNA can also be prepared using a large-scale PCR method as detailed in a recent protocol (Nodelman et al., 2020).
Reconstitution of the nucleosome essentially follows a previous protocol (Dyer et al., 2004).

Preparation of recombinant H1 and its chaperone ProTa
Timing: 3-4 days H1 and ProTa genes were codon-optimized, commercially synthesized and cloned into the pET42b or pET28b vector using NdeI and BamHI restriction sites. In order to purify the full-length recombinant protein using Ni-NTA beads, we added an in-frame his6 tag at the C-terminus of H1 and ProTa.

Expression of H1 and ProTa in bacterial cell
17. Transform 1 mL of pET42b-H1 or pET28-ProTa plasmid into 20 mL BL21(DE3) RIPL cells following the manufacturer's protocol. Spread $10 mL of transformed cells onto an LB agar plate containing 50 ug/mL kanamycin. Incubate the plate in a 37 C incubator for $18 h. 18. Inoculate a single colony into 10 mL LB medium containing 50 mg/mL kanamycin and grow in a 37 C incubator shaker at 220 rpm for 16 h. 19. Dilute the 10 mL cell culture into 1 L LB medium containing 50 mg/mL kanamycin. Continue to grow until the OD 600 reaches 0.6-0.8. Add 0.5 mM IPTG to induce protein expression for 3 h. 20. Harvest the cells by centrifugation at 3000xg, 10 min at 4 C in a Beckman Coulter Avanti J-20I centrifuge. Store the cell pellet at À80 C until the next step. Combine the fractions that contain protein, inject into a protein RP HPLC column, and elute the protein by linearly increasing the acetonitrile concentration from 10% to 60% using a Waters HPLC system. 27. Freeze the elution peak containing pure protein on dry ice and lyophilize the protein using a freeze dryer.

OPEN ACCESS
Note: Lyophilized protein powder is stable for years and can be stored either at 22 C (avoid direct light) or at À80 C.
Alternatives: If an HPLC instrument is not available, proteins from step 25 can be buffer exchanged into low salt buffer (50 mM sodium phosphate, 6 M urea, 0.15 M NaCl, pH 8) by dialysis and injected into a Hitrap SP column (for H1) or Hitrap Q column (for ProTa). Elute protein by linearly increasing the salt concentration from 0.15 to 0.6 M using an AKTA FPLC. Finally, remove all the buffer and salt components by thoroughly dialyzing against DI water, then lyophilize and store H1 and ProTa as described above.

Lysis buffer
Cell lysis buffer contains 50 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM EDTA. Add the reagents below to 800 mL DI water: Wash buffer Inclusion body wash buffer contains 50 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100. Add the reagents below to 800 mL DI water: Adjust pH to 7.5 at 25 C, add DI water up to 1 L. The buffer can be stored at 4 C for one month.

Refolding buffer
Refolding buffer contains 100 mM Tris-HCl, pH 9.5, 0.5 M arginine, 1 mM EDTA and 551 mg/L oxidized glutathione. Add the reagents below to 800 mL DI water: Note: Oxidized glutathione powder should be added to the buffer just before use.

Dialysis buffer
Dialysis buffer contains 20 mM Tris-HCl, pH 7.4, 100 mM urea. Add the reagents below to 4 L DI water: Note: 30 g urea should be added to the buffer just before use. Adjust pH to 7.5 at 25 C, add DI water up to 1 L. The buffer can be stored at 4 C for one month. Adjust pH to 7.4 at 25 C, add DI water up to 4.5 L. The buffer should be chilled to 4 C one day before use.

Refolding and purification of scFv from inclusion body
Timing: 9-10 days

Preparation of scFv from the inclusion body
1. Transform 1 ml pETHis6TEV-scFv plasmid into 20 ml BL21(DE3) RIPL cells following the manufacturer's protocol. Spread $20 mL transformed cells onto an LB agar plate containing 100 mg/mL ampicillin. Incubate the plate at 37 C for 18 h. 2. Inoculate a single colony into 10 mL LB medium containing 100 mg/mL ampicillin and grow in a 37 C incubator shaker at 220 rpm for 14 h. 3. Dilute the 10 mL LB cell culture into 1 L of 2x YT medium containing 100 mg/mL ampicillin.
Continue to grow until the OD 600 of the cell culture reaches 0.8. 4. Cool the cell culture and the incubator shaker to 25 C. Induce protein expression by adding 0.5 mM IPTG and continue to grow the cells in the 25 C incubator shaker at 220 rpm for 16 h. 5. Harvest the cells by centrifugation at 3000xg, 10 min at 4 C in a Beckman Coulter Avanti J-20I centrifuge. Resuspend the cells in 35 mL Lysis buffer (50 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM EDTA). Add 0.5 mL 50 mg/mL lysozyme stock solution and freeze the cells on dry ice. Inject the sample into a 5 mL pre-equilibrated Histrap column, wash with 5 volumes of Histrap wash buffer. Elute by linearly increasing the imidazole concentration from 40 mM to 250 mM over 20 column volumes using an AKTA FPLC. 23. Collect fractions from the main elution peak (appears around 80 mL). Check each fraction on an SDS PAGE gel (Figure 4, lanes 3-9). The protein yield after Histrap chromatography is $5 -10 mg Combine the fractions containing scFv and concentrate them to around 4 mL.
CRITICAL: Do not over concentrate the scFv as it will aggregate at concentration above 5 mg/mL.

24.
Equilibrate the Superdex S75 10/600 column with Gel-Filtration buffer A (20 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA) for 2 column volumes. 25. Inject 2 mL of the scFv sample from step 23 to the Superdex S75 10/600 column with a flow rate of 1 mL/min. The scFv with correct disulfide bonds appears at around 65 mL. Check each fraction on an SDS PAGE gel (Figure 4, lanes 10-13). Troubleshooting 2 26. Concentrate the peak fractions to $1.5 mL volume using a 10 kDa cut-off Amicon concentration device. Keep the concentration to around 5 uM, as a higher concentration can lead to aggregation and loss of scFv. The protein yield after S75 chromatography is $3 -5 mg. Store scFv in a 4 C refrigerator. Troubleshooting 3

OPEN ACCESS
To prepare the scFv stabilized chromatosome, we first do small volume titration of 197 bp nucleosome with linker histone in the presence of chaperone ProTa and analyze each titration on a native PAGE gel. After the best ratio is determined, we then do large scale preparation of chromatosomes, and add scFv to the chromatosome sample before preparation of the cryo-EM grids. Adjust the final volume of the binding reaction to 10 mL and incubate at 25 C for 15 min. 31. Load 5 mL of each reaction on a 4.8% native PAGE gel, run in 0.2x TBE buffer at 100 V for 120 min at 4 C. 32. Stain the gel with Midori Green Advance stain, visualize the gel using a ChemiDoc gel imaging system ( Figure 5). 33. Determine the best ratio for the nucleosome and H1. Use that ratio to make a large volume (>100 mL) of chromatosome by titration of the nucleosome with H1, in the presence of ProTa. Troubleshooting 4 CRITICAL: Mixing H1 and nucleosome at a high concentration (>100 uM) can cause aggregation. To prevent sample loss, dilute both the nucleosome and H1 to a concentration below 10 uM before titration or mixing.
Preparation of scFv stabilized chromatosomes 34. Dilute scFv with TEN10 buffer to a concentration of around 0.5 uM, add 3x molar ratio of scFv to the chromatosome sample and incubate on ice for 15 min. 35. To remove extra scFv and ProTa, concentrate the complex to around 500 mL and inject onto a Superose 6 10/300GL column pre-equilibrated with TEN10 buffer. Run at 0.5 mL/min using an AKTA FPLC. Collect the peak fraction and concentrate to $100 mL using an Amicon Ultra 30K MWCO centrifugal filter unit.
Vitrification of scFv stabilized chromatosome and screen grids using cryo-TEM 36. Measure the concentration of the scFv-bound chromatosome. Adjust the concentration to 4-5 uM with TEN10 buffer for sample vitrification. 37. Prepare liquid nitrogen cooled ethane and set the operation parameters of the Vitrobot Mark IV (Thermo Fisher Scientific) to 4 C, 100% humidity, 3 s blotting time. 38. Pretreat Quantifoil 1.2/1.3 holy carbon copper grids at 15 mA for 60 s using an easiGlow Glow Discharge Cleaning System. 39. Apply 3 mL of the chromatosome sample to the grid, blot and vitrify the grid using the Vitrobot.
Transfer and store the grid in a liquid nitrogen tank. 40. For the latest generation of electron microscope with an autoloading system, assemble the grid with a clip ring and a C-shape spring in the ''auto-grid'' cartridge system. 41. Insert the grid into the electron microscope by the autoloader, screen the grids using SerialEM (Mastronarde, 2005). Troubleshooting 5 Optional: For a traditional electron microscope with side-entry, chill the workstation with liquid nitrogen, carefully transfer the grid to the tip the cryo-holder and secure the grid with the clip ring. With the grid covered by the shutter, promptly insert the holder into the microscope from the side-entry. The grid is then ready for screening.

EXPECTED OUTCOMES
Sample dissociation during the blotting and vitrification process hinders the study of many protein complexes by cryo-EM. In this protocol, we use scFv to stabilize the chromatosome (a nucleosome bound to a histone H1), which prevents chromatosomes from being absorbed to the water-air interface. Thus the whole complex remains intact in the vitrified ice. Our protocol does not rely on chemical crosslinking that many groups use to stabilize the nucleosome or its complexes. It can be broadly used to study the nucleosome with a natural DNA sequence or any nucleosome protein complex, provided that the scFv does not interfere with protein nucleosome interaction. Typical cryo-EM micrograph of the scFv stabilized chromatosome complex is shown in Figure 6.

LIMITATIONS
Our protocol has the following limitations: scFv binds to the nucleosome acidic patch, which limits its application to nucleosome complexes in which proteins also interact with the acidic patch of the nucleosome. The procedure for purification of scFv may not apply to other scFv, as the charge property of each scFv may be different. For example, if the scFv has a very low isoelectric point (PI), then one should consider using anion resins instead of cation resins for ion exchange purification.

TROUBLESHOOTING
Problem 1 scFv does not bind to the SP beads.

Potential solution
Carefully check the pH of the scFv refolding solution, make sure the pH is around or slightly below 7.8. Purified scFv has two bands on the SDS PAGE gel.

Potential solution
Folded scFv with correct disulfide bonds runs slightly slower than misfolded scFv on the SDS PAGE gel (Figure 4). Try to rerun the Superdex S75 column and collect smaller fractions to better separate the folded scFv from the misfolded scFv.
Problem 3 scFv yield is too low.

Potential solution
Multiple factors could affect the yield of scFv: At the denaturation step, make sure that the cysteines in the scFv are in reduced form by adding 1,4-DTE powder fresh to the denaturation buffer. DTE should be stored at À20 C as it is prone to oxidation at higher temperatures.
At the refolding step, add oxidized glutathione right before adding the denatured scFv solution. Limit the refolding time to at least 36 h, as refolding time that is too short or too long (> 1 week) will result in a low yield of the correctly refolded scFv.
At the purification step, note that scFv is not stable at 25 C, especially when it is not pure. Try to do all the purification steps at 4 C and chill all the buffers before use.
At the concentrating and storage steps, do not over-concentrate scFv, as it is not stable at concentrations that are higher than 10 uM. It is best to store it at a concentration of $ 5 uM in physiological buffer conditions and at 4 C. Freeze and thaw are not recommended.

Problem 4
Loss of chromatosome during large scale preparation.

Potential solution
The ratio of H1 to nucleosome should be carefully determined from the 4.8% native PAGE gel ( Figure 5). When the number of H1 molecules is more than the nucleosomes, it binds to nucleosomes nonspecifically and causes aggregations. Dilute both the H1 and nucleosome to a concentration of 10 uM or less using TEN10 buffer. Generally, a step-by-step titration of nucleosome with H1 will help.

Problem 5
Cryo-EM screen shows complex dissociation, even for scFv stabilized nucleosome or chromatosomes

Potential solution
This happens when the vitrified ice is super thin. Try different blotting times and use a different kind of grid such as a Lacey grid.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yawen Bai (baiyaw@mail.nih.gov).

Materials availability
Unique and stable reagents generated in this study are available upon request.

Data and code availability
This protocol did not generate datasets or code.