Superose 6 column

The purified RsbR and RsbS were combined in a 1:2 ratio of RsbR to RsbS before being subjected to a gel filtration. The Superose 6 column was pre-equilibrated in 20 mM Tris pH 8.5, 1 mM DTT. Only fractions containing RsbR-RsbS proteins in the void volume fractions were pooled and concentrated by centrifugation using a 30 kDa molecular weight cutoff centrifugal filter (Vivaspin). Purified RsbR:RsbS complex was combined with an excess of RsbT and incubated at 4 °C for 4 h followed by gel filtration using a Superose 6 column (GE Healthcare) pre-equilibrated in freshly prepared 20 mM Tris HCl pH 8.5, 150 mM NaCl, 1 mM adenosine diphosphate, 1 mM DTT. Only those fractions containing RsbR-RsbS-RsbT proteins were collected.

Large-scale expression of recombinant human HDACs was carried out
as described previously.^{32 (link),37 (link)} Briefly, HEK293/T17
cells were transiently transfected with expression pMM222 plasmids
comprising N-terminally tagged (TwinStrep-FLAG-HALO tag; Figure S1) HDAC genes using linear poly(ethyleneimine)
(PEI, Polysciences Inc.). Cells were harvested three days post-transfection,
and cell pellets were resuspended in the lysis buffer (100 mM Tris-HCl,
10 mM NaCl, 5 mM KCl, 2 mM MgCl₂, and 10% glycerol at pH
8.0). Cells were disrupted by sonication, cell lysates were cleared
by centrifugation, and soluble fusion proteins were first purified
by Streptactin affinity chromatography (IBA). For all HDACs but HDACs
1, 10, and 11, the N-terminal tag was cleaved off overnight by the
addition of TEV protease (10:1 HDAC/TEV ratio). Size-exclusion chromatography
on a Superose 6 column (GE Healthcare Bio-Sciences; running buffer
30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP)
was used as the final purification step for all HDAC constructs.

Wild type and mutant MTA1 constructs were cloned into pcDNA3 vectors containing an amino terminal His₁₀-Flag₃ purification tag and a TEV protease cleavage site. Full length HDAC1 (residues 1–482), RBBP4 (residues 1–425) and MBD2 (residues 145–411) were cloned without affinity tags into the same vectors. Protein was expressed in HEK293F suspension-grown cells (Invitrogen) using polyethylenimine (PEI; Sigma) as a transfection reagent and harvested after 48 h as previously described (15 (link),30 ). Cells were lysed in 50 mM Tris–HCl (pH 7.5), 100 mM potassium acetate, 10% (v/v) glycerol, 0.3% (v/v) Triton X-100 and Roche Protease Inhibitor (buffer A). The lysate was clarified by centrifugation and applied to FLAG resin (Sigma) for 30 min and washed three times with 50 mM Tris–HCl (pH 7.5), 100 mM potassium acetate and 5% (v/v) glycerol (buffer B). The protein was treated with RNase A in buffer B for 1 h, washed twice more with 25 mM Tris–HCl (pH 7.5), 75 mM potassium acetate and 0.5 mM TCEP (buffer C), before being eluted with TEV protease overnight. The protein complexes were purified further by gel filtration using a Superose 6 column (GE Healthcare, UK).

SynNF1 was expressed and purified as described^{33 (link),40 (link)}. For the production of synNF1eGFP Sf21 cells were infected with saturating amounts (multiplicity of infection (MOI) 1–2) of recombinant synNF1 or synNF1eGFP expressing baculoviruses, harvested via centrifugation after 72 h post-infection and stored at −80 °C. All subsequent steps were carried out at 4 °C. The cell pellet was resuspended in lysis buffer [50 mM HEPES pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM TCEP, 10 mM imidazole] supplemented with cOmplete, EDTA-free protease inhibitor mix (Roche) and cells were lysed by a freeze thaw cycle in liquid nitrogen. Recombinant neurofibromin was purified from the soluble fraction via immobilized metal ion affinity chromatography (IMAC) using HisTrap FF columns (GE Healthcare) followed by a size exclusion chromatography (SEC) on a Superose 6 column (GE Healthcare) in 50 mM HEPES pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM TCEP. SEC fractions containing synNF1 or synNF1eGFP were collected and concentrated using Vivaspin Centrifugal Concentrators with a cutoff of 100 kDa (Santorius). The purified protein was flash frozen in liquid nitrogen and stored at −80 °C.

SpoIIE was expressed as an N-terminal Sumo-6His fusion in BL21(DE3) cells following overnight induction with 0.5 mM IPTG at 14°C. Cells were lysed in 50 mM Tris pH 8.5, 200 mM NaCl, 1mM beta-mercaptoethanol and purified using HisTrap columns (GE Healthcare, Pittsburg, PA) eluting with a gradient of imidazole. The Sumo-6His tag was removed by cleavage with Ulp1 (Sumo Protease) followed by Ni-NTA subtraction. For velocity analytical ultracentrifugation, SpoIIE was dialyzed to 20 mM Tris pH8.5, 100 mM NaCl, 2 mM DTT overnight and data was collected at 280 nM spinning at 20,000 RPM at The Biophysical Instrumentation Facility (NSF-0070319) at MIT. Data were fit to a continuous model using SedFit (Schuck, 2000 (link)). Gel filtration was conducted on a 24 ml Superose 6 column (GE Healthcare, Pittsburg, PA), loading 100 µl of 1 µM SpoIIE. Phosphatase assays of soluble fragments of SpoIIE lacking the transmembrane domain (SpoIIE^320-827 and SpoIIE^{320-827, V697A}) were performed using 32P phosphorylated SpoIIAA (phosphorylated by purified SpoIIAB) as a substrate. Multiple turnover reactions were performed with 0.05 µM SpoIIE and varying concentrations of SpoIIAA-P as indicated. Dephosphorylation of SpoIIAA was detected by TLC chromatography on PEI-Cellulose plates developed in 1 M LiCl, 0.8 M Acetic acid.

Trimer (500 μg) was incubated with 10-fold molar excess of 43A2 Fab overnight. The complex was then purified over Superose 6 column (GE Healthcare), concentrated to 1.5 mg/ml, and mixed with Lauryl Maltose Neopentyl Glycol (Anatrace) before deposition onto 2/2 Quantifoil grids (EMS) that were glow-discharged for 10 s, directly preceding the deposition in a Vitrobot (Thermo Fisher Scientific). Once sample was deposited, the grids were blotted and plunged into liquid ethane using the Vitrobot to immobilize the particles in vitreous ice. Using Leginon image acquisition software, we collected 1366 micrographs at a nominal magnification of ×29,000 with a Gatan K2 summit detector mounted on a Titan Krios set to 300 kV set to counting mode for the data collection (44 (link)). The dose rate was ~4.78 e⁻/pix per second with frame exposure of 250 ms, with a total exposure time and dose of 14 s and 60 e⁻/Ų, respectively. MotionCor2 was used for frame alignment, and CTF models were determined using GCTF (49 (link)). DoG Picker was used to pick 455,207 particles, which were then extracted and subsequently 2D-classified in cryoSPARC (45 (link), 50 (link)). Selected 2D classes amounting to 85,841 particles were then fed into the 3D homogeneous refinement algorithm using C3 symmetry, resulting in final resolution of ~3.52 Å.