Protein expression, mutagenesis and purification
Table of Contents
Xenopus laevis histones for nucleosome assembly were overexpressed in the Escherichia coli BL21(DE3) pLysS strain and purified from inclusion body as previously described48.
The cells were grown in LB medium at 37 °C and induced with 1 mM IPTG when OD600 reached 0.6. After 3 h of expression, the cells were pelleted down, resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF) and frozen. Later, the frozen cells were thawed and sonicated. The pellet containing inclusion bodies was recovered by centrifugation at 5,000 rpm for 20 min at 4 °C. The inclusion body pellet was washed three times with lysis buffer containing 1% Triton X-100, followed by two washes with lysis buffer without Triton X-100.
Each histone protein was extracted from the purified inclusion body pellet in a buffer containing 50 mM Tris (pH 7.5), 2 M NaCl, 6 M guanidine hydrochloride and 1 mM DTT for overnight at room temperature. Any insoluble components were removed by centrifugation. Proteins making histone pairs (H2A–H2B and H3–H4) were combined in equimolar ratios and dialysed two times in 1 l of refolding buffer (25 mM HEPES/NaOH (pH 7.5), 2 M NaCl and 1 mM DTT) at 4 °C. Any precipitate was removed by centrifugation for 20 min at 13,000 rpm at 4 °C. The soluble histone pairs were further purified via cation-exchange chromatography in batch (SP Sepharose Fast Flow resin). The samples were diluted fourfold with buffer without salt (25 mM HEPES/NaOH (pH 7.5) and 1 mM DTT) and bound to the resin for 30 min. The resin was extensively washed with 500 mM salt buffer in batch (25 mM HEPES/NaOH (pH 7.5), 500 mM NaCl and 1 mM DTT) and loaded onto a disposable column. On the column, the resin was washed, and pure proteins were eluted with 25 mM HEPES/NaOH (pH 7.5), 2 M NaCl and 1 mM DTT. Soluble histone pairs were concentrated and purified on a Superdex S200 size-exclusion column (GE) equilibrated in 25 mM HEPES/NaOH (pH 7.5), 2 M NaCl and 1 mM DTT. Clean protein fractions were pooled, concentrated and flash frozen.
For cryo-EM grid freezing of ‘assembly 1’ (see below), commercially available OCT4 from Abcam (ab 134876) was used. The protein (approximately 52 kDa) was fused with the herpes simplex virus VP16 transactivation domain at the N terminus and a 11R tag at the C terminus. For the ‘assembly 2’ for cryo-EM and all the other assays, His-tagged OCT4 (approximately 39 kDa) was expressed in a pET28 vector and purified under denaturing conditions from inclusion body using Talon affinity resins. To refold the OCT4 protein, the first overnight dialysis was carried out in 2 M urea, 50 mM HEPES (pH 7.5), 250 mM NaCl, 50 mM l-arginine and 2 mM DTT. Then, the second and third dialyses were carried out for 1 h in a buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl and 1 mM DTT.
All the OCT4 variants were generated using the inverse PCR strategy. Oligo primers used for mutagenesis were purchased from Integrated DNA Technology and are listed in the Supplementary Table 1. The inverse PCRs were set up in a total volume of 25 μl. After amplification, 10 μl of purified PCR product was incubated with 5 U of T4 PNK in 20 μl of 1× T4 DNA ligase buffer for 1 h at 37 °C. Of T4 DNA ligase, 200 U was added to the reaction and incubated for 1 h at room temperature. Finally, 10 U of Dpn I was added to the reaction and incubated for 1 h at 37 °C. From this mixture, 5 μl was used to transform the competent XL1-Blue E. coli cells. The clones were selected on kanamycin plates and were subsequently confirmed by sequencing.
Histone octamer assembly and purification
Histone octamer purification was done using the standard protocol48,49. In brief, a 2.5-fold molar excess of the H2A–H2B dimer was mixed with the H3–H4 tetramer in the presence of buffer containing 2 M NaCl (25 mM HEPES (pH 7.5), 2 M NaCl and 1 mM DTT). After overnight incubation at 4 °C, the assembled octamer was separated from excess dimer using a Superdex S200 Increase 10/300 GL column on an AKTA FPLC system. The fractions were analysed on SDS–PAGE, pooled and concentrated for final nucleosome assembly.
LIN28B 182-bp DNA amplification
A custom synthesized (Integrated DNA Technology) 162-bp LIN28 genomic DNA8 was cloned into the pDuet plasmid. To make the longer 182-bp LIN28B DNA fragment by PCR, two primers were designed so that each contained an extra 10 bases from the flanking genomic region of the canonical 162-bp LIN28 fragment used in previous studies8. The DNA sequence for the 182-bp extended DNA used in this study is shown in Supplementary Table 1.
Mutant LIN28B DNA
Custom synthesized 182-bp LIN28B DNA was purchased from Integrated DNA Technology with the following mutations in the three OCT4-binding sites:
LIN28B-1M: ATT AAC AT – GCGTCGAT
LIN28B-2M: ATT AAC AT – GCG GCT AT
LIN28B-3M: ATG CTG AAT – GCG GGT AA
The fragments were later PCR amplified to generate DNA for nucleosome assemblies.
OCT4-binding DNA sequences from the human genome
The 186-bp nMATN1 sequence was selected from the human genome (https://www.ncbi.nlm.nih.gov/genome/gdv) from the position GRCh38:1:30216402:30217024:1 on chromosome 1 (ref. 3). The DNA fragment was selected based on the presence of the following OCT4 motifs: ATGCTAAT, ATTAGCAT, ATTAACAT or ATGTTAAT. The 186-bp nMATN1 sequence is shown in Supplementary Table 1.
Nucleosome assembly
Nucleosome assembly was carried out using a ‘double bag’ dialysis method as previously described50,51. The histone octamer and nucleosomal DNA fragment were mixed in equimolar ratios in a buffer containing 50 mM HEPES (pH 7.5), 2 M NaCl and 2 mM DTT. The mixture was placed into a dialysis button made with a membrane with a cut-off of 3.5 kDa. The dialysis button was placed inside a dialysis bag (6–8-kDa cut-off membrane) filled with 50 ml of buffer containing 25 mM HEPES (pH 7.5), 2 M NaCl and 1 mM DTT. The dialysis bag was immersed into 1 l of buffer containing 25 mM HEPES (pH 7.5), 1 M NaCl and 1 mM DTT, and dialyzed overnight at 4 °C. The next day, the buffer was changed to 1 l of a buffer with 25 mM HEPES (pH 7.5) and 1 mM DTT, and dialysis was continued for 6–8 h. In the last step, the dialysis button was removed from the dialysis bag and dialysed overnight into a fresh buffer without any salt (50 mM HEPES (pH 7.5) and 1 mM DTT). The nucleosome assemblies were assessed on a 6% native PAGE using SYBR Gold staining.
Assembly of modified nucleosomes
H3K27ac nucleosomes were assembled using the LIN28B DNA (unlabelled or Cy5-labelled) and histone octamer with H3K27ac modification (custom purchased from Epicypher). For H3K27me3 nucleosomes, the H3K27C-mutant histone was generated using site-directed mutagenesis and later expressed and purified from E. coli. The H3K27C-mutant histone thus obtained was trimethylated using the MLA protocol52 and was purified using a PD10 column. This trimethylated H3 was used with other histones for octamer assembly. The purified H3K27me3 octamer was mixed with LIN28B and nMATN1 DNA for the assembly of H3K27me3 nucleosomes.
Nucleosome array assembly
A 1,022-bp genomic region from the LIN28 genomic site was synthesized by DNA synthesis (Codex, Protein Technology Center, St Jude Children’s Research Hospital). For nucleosome array reconstitution, the DNA fragment was amplified to a larger scale by PCR. For the assembly, the DNA and histone octamer were mixed in a 1:5 ratio.
Nucleosome array reconstitution was carried out using the double bag dialysis salt dilution method described above (see ‘Nucleosome assembly’).
The synthesized genomic DNA sequence used for array assembly (the LIN28B 182-bp region shown in bold) is shown in Supplementary Table 1.
Assembly of the nucleosome–OCT4 complex for cryo-EM grid freezing
The LIN28B complex
Equimolar mixture of histone octamer and LIN28B DNA (2 µM each) were mixed with 1 µM of OCT4 (ab134876, Abcam) and 3 µM of SOX2 (50 mM HEPES (pH 7.5), 2 M NaCl, 20% glycerol and 5 mM DTT). The assembly was carried out with four steps of buffer changes over 72 h. The buffer changes were carried out to dilute out the salt concentration from 2 M starting concentration to a final solvent condition of no salt. The three buffers, used for the assembly dialysis, contained 50 mM HEPES (pH 7.5), 2 mM DTT and varying NaCl concentrations of 2 M, 1 M and 0, respectively. After the assembly, the samples were centrifuged at 13,000 rpm for 10 min at 4 °C to remove any precipitates. Following this, the sample was concentrated using a 10 kDa Centricon to the concentrations needed for cryo-EM grid freezing (0.5–1 µg µl−1).
The assemblies were checked on 6% native gels followed by native western blot analysis. For the detection of nucleosomes, OCT4 and SOX2, anti-H3, anti-OCT4 and anti-His antibodies were used, respectively (see the section ‘Western blot detection’ below).
The nMATN1 complex
For the OCT4 bound to the nMATN1 nucleosome, 1 µM of pre-assembled nucleosomes were mixed with 2 µM of His-tagged OCT4 (see above) and incubated at room temperature for 30 min. The sample was then transferred to ice until grid freezing.
Restriction enzyme Mnl I digestion assays
For digestion of LIN28B nucleosomes, different dilutions of Mnl I (NEB) were made in the 1× CutSmart buffer (NEB). The digestion was carried out for 30 min at 25 °C. For the experiments involving OCT4 and OCT4 variants, the protein was incubated with nucleosomes at 25 °C for 5 min before the addition of Mnl I. After the addition of Mnl I, the samples were kept at 25 °C for 30 min. After the digestion, the samples were run on a 6% polyacrylamide gel to separate all the products and then imaged by SYBR Gold staining on a Typhoon scanner.
Magnesium precipitation assay
LIN28B nucleosome samples were incubated in varying MgCl2 concentrations for 10 min at 25 °C. The precipitated nucleosomes were separated from soluble nucleosomes by spinning at 10,000 rpm for 10 min at 25 °C. The same procedure was followed for nucleosome samples containing wild-type OCT4 and other variants. However, for the experiments done in the presence of OCT4 and OCT4 variants, the nucleosomes were first mixed with fivefold molar excess of OCT4 (or OCT variants) and kept at 25 °C for 5 min before any MgCl2 addition.
Binding assays
The binding assays with OCT4 were performed at 25 °C in 50 mM HEPES (pH 7.5), 200 mM KCl, 1 mM DTT and 0.005% NP-40. The binding assays involving both OCT4 and SOX2 were performed in 50 mM HEPES (pH 7.5) and 1 mM DTT. Typically, 20–40 nM of nucleosome was incubated with different amounts of proteins (OCT4, OCT4 variants and SOX2). For the OCT4-binding experiments, the reaction was incubated for 10 min. For binding involving both OCT4 and SOX2, the reaction was incubated for 10 min after the addition of OCT4, following which SOX2 was added and kept for an additional 5 min. The bound and unbound species were separated on a 5% or 6% native polyacrylamide gel and imaged for Cy5 fluorescence using a Typhoon scanner. In experiments with nucleosomes without Cy5 label, SYBR Gold staining was used to visualize the gels.
Analysis of gels
All the gels were analysed using Quantity One Basic version (Bio-Rad). The data were exported and analysed or plotted using Open Office Calc. All the bands were selected using boxes of the same size: 24 mm2 for input nucleosome and 8 mm2 for all other bands. The background correction was done separately for bands from each lane using boxes of identical size in the same lane.
Analysis of Mnl I digestion of nucleosomes
In the nucleosome-only experiment, after background correction, the signal from the nucleosome band from each concentration point was normalized to the signal from the nucleosome lane in the 0 Mnl I lane. For Mnl I digestion in the presence of OCT4 or its variants, the signal of the OCT4-bound band from each of the Mnl I concentration was background corrected and then normalized to the signal of the OCT4-bound band from the 0 Mnl I lane.
Analysis of the Mg2+ precipitation assays
The relative compaction was calculated as the fraction of the precipitated nucleosomes. For this, the following formula was used: relative compaction = S0 – Sobs. S0 is the signal of the nucleosome band at the 0 Mg2+ concentration normalized to 1, and Sobs is the signal of all the soluble nucleosome bands normalized to the signal of nucleosomes at the 0 Mg2+ concentration. For precipitation experiments in the presence of OCT4 or its variants, the signals from both the bound and the unbound nucleosomal species were summed to calculate the soluble nucleosomes.
Analysis of OCT4 binding to wild-type LIN28B versus the LIN28B-1M mutant
For binding to wild-type LIN28B nucleosomes, all bands were normalized to input nucleosome. For comparison, we used the following equation: binding to OBS2/3 = ‘2-OCT4’/(‘1-OCT4’ + ‘2-OCT4’), where ‘2-OCT4’ represents a nucleosome with two OCT4 bound (OBS1 + OBS2/3), and ‘1-OCT4’ is a nucleosome with one OCT4 bound (OBS1). ‘1-OCT4’ + ‘2-OCT4’ represents input OCT4 bound to OBS1 nucleosomes, which are substrates for binding of the second OCT4. For binding to LIN28B-1M nucleosomes, we used the following equation: binding to OBS2/3 = ‘1-OCT4’/nucleosome, where nucleosome represents input nucleosomes.
Analysis of SOX2 binding to wild-type LIN28B
Binding of SOX2 to OCT-bound nucleosome was calculated as the fraction of SOX2 bound to the OCT4-bound LIN28B nucleosome: SOX2 = ‘1-OCT4–SOX2’/(‘OCT4’ + ‘1-OCT4–SOX2’), where ‘1-OCT4–SOX2’ represent nucleosomes with both OCT4 and SOX2 bound, and OCT4 represents OCT4-bound nucleosomes. ‘1-OCT4’ + ‘1-OCT4–SOX2’ represents input OCT4-bound nucleosomes, which are substrates for binding of the SOX2 to OCT4-bound nucleosomes. Binding of SOX2 to the LIN28B nucleosome is shown as a fraction of free LIN28B nucleosomes: SOX2 = SOX2/nucleosome), where SOX2 represents SOX2-bound nucleosomes and nucleosome represents input nucleosomes.
Analysis of OCT4 binding to unmodified, H3K27ac and H3K27me3 nucleosomes
For binding to modified nucleosomes, we used the following equations: 1st OCT4 = (‘1-OCT4’ + ‘2-OCT4’ + ‘3-OCT4’)/input nucleosome; 2nd OCT4 = (‘2-OCT4’ + ‘3-OCT4’)/(input nucleosome); and 3rd OCT4 = ‘3-OCT4’/(input nucleosome), where 1st, 2nd or 3rd OCT4 indicates binding of the 1st, 2nd or 3rd molecule of OCT4, respectively, ‘1-OCT4’ is a nucleosome with one OCT4 bound, ‘2-OCT4’ is a nucleosome with two OCT4 bound, and ‘3-OCT4’ is a nucleosome with three OCT4 bound. The quantification is shown as a ratio of modified nucleosomes to unmodified nucleosomes (1st OCT4 modified/1st OCT4 unmodified).
Analysis of SOX2 binding to H3K27ac nucleosomes
For binding to modified nucleosomes, we used the following equation: ‘SOX2–OCT4’ = ‘1-OCT4–SOX2’/(‘1-OCT4–SOX2’ + ‘1-OCT4’), where ‘SOX2–OCT4’ represents SOX2 binding to nucleosome with one OCT4 bound, ‘1-OCT4’ represents a nucleosome with one OCT4 bound, and ‘1-OCT4–SOX2’ is a nucleosome with OCT4 and SOX2 bound. The quantification is shown as a ratio of modified nucleosomes to unmodified nucleosomes (‘SOX2–OCT4’ modified/‘SOX2–OCT4’ unmodified).
Western blot detection
SDS–PAGE gels or native PAGE gels were transferred to a PVDF membrane and blocked in TBST (50 mM Tris/HCl (pH 7.5), 150 mM NaCl and 0.1% Tween-20) containing 5% milk for 1 h. Membranes were then incubated in primary antibody in TBST containing 5% milk for 1 h at room temperature. The membranes were washed three times for 5 min with TBST and incubated in secondary antibody for 1 h at room temperature. Membranes were washed three times (approximately 5 min each) with TBST before chemiluminescent detection. The following antibodies were used: anti-OCT4 antibody (1:2,000 dilution; ab109183, Abcam), horseradish peroxidase-conjugated anti-His antibody (1:3,000 dilution; R931-25, Invitrogen–Thermo Fisher), anti-H3 antibody (1:3,000 dilution; ab1791, Abcam) and anti-SOX2 antibody (1:2,000 dilution; ab92494, Abcam), horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000 dilution; 170-6515, Bio-Rad).
MNase-seq
OCT4 was bound to unmodified, H3K27ac or H3K27me3 nucleosomes with LIN28B or nMATN1 DNA (20 mM HEPES (pH 7.5), 50 mM KCl, 2.5 mM MgCl2 and 5 mM CaCl2) and digested by MNase (NEB) for 5 min at 25 °C. MNase digestion was terminated by 50 mM EDTA. Cleaved nucleosome was subjected to phenol/chloroform extraction followed by ethanol precipitation of nuclesomal DNA and used for library preparation. The sequencing library was prepared using the NEBNext Ultra II DNA Library Prep Kit following the manufacturer’s manual. Amplification of the library for Illumina sequencing was performed by PCR using NEBNext Multiplex Oligos for the Illumina kit. Sequencing was pair ended with 100-bp length. Paired reads were merged and filtered by the length of reads between 144 bp and 146 bp and mapped to the LIN28B or nMATN1 sequence with Qiagen CLC genomics Workbench 20 software.
MiDAC purification
MiDAC was purified from 1.25 l of adherent Flp-In 293 T-REx (R78007, Thermo Fisher Scientific) cell lines stably transformed with the Flp-In expression vector carrying FLAG-ELMSAN1/MIDEAS. The cells were grown in DMEM media (Gibco) supplemented with 10% FBS, 100 µg ml−1 hygromycin and induced for 24 h with 1 µg ml−1 doxycycline (Thermo Fisher Scientific). Cells were harvested and lysed using the classical Dignam protocol53. The complex was isolated from the nuclear fraction using anti-FLAG M2 beads from Sigma-Aldrich. The nuclear fraction was mixed with washed FLAG M2 beads and incubated overnight at 4 °C. The next day, the beads were washed with wash buffer (20 mM HEPES (pH 7.9), 300 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 0.5 mM DTT and protease inhibitors (Sigma)) four times. The complex was eluted from the beads in the elution buffer (20 mM HEPES (pH 7.9), 100 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT and protease inhibitors (Sigma)) after 30 min of incubation at 4 °C. This complex was flash frozen in liquid nitrogen and stored at −80 °C.
Deacetylation of H3K27ac nucleosomes
H3K27ac nucleosomes were deacetylated by the human MiDAC deacetylase complex. The deacetylation reaction was carried out for 18 h at 25 °C in the following buffer: 50 mM HEPES (pH 7.5), 100 mM KCl and 0.2 mg ml−1 BSA. A control parallel reaction containing H3K27ac nucleosomes, but no MiDAC, was also carried out under identical conditions. The extent of deacetylation was confirmed by western blot using anti-H3K27ac antibody.
Negative-stain EM
For the experiment looking at array compaction, 20 nM of the LIN28 array was mixed with MgCl2 to a final [Mg2+] of 3 mM. For analysis of the effect of wild-type and ΔN OCT4 proteins, 70 nM (wild type) and 100 nM (ΔN) proteins were used with the mixture of array and MgCl2.
After approximately 10–15 min of incubation at 25 °C, 3 µl of the sample was added to Lassey carbon or quantifoil grids for 1 min, blotted dry and stained. For staining, four separate drops (approximately 40 µl) of uranyl acetate or uranyl formate were added to a parafilm strip. The grid was briefly brought into contact with the stain for the first three drops before quick blotting. The last drop of stain was kept in contact with the grid for 1 min before the final blot drying.
The dried grids were imaged on a Talos L 120C microscope (Thermo Fisher Scientific) at the cryo-EM facility at St Jude Children’s Research Hospital. Several images were acquired at ×73,000–92,000 magnification from regions showing good particle distribution. Specifically, a magnification of ×73,000 was used for experiments involving Mg2+ compacted arrays in the absence or presence of OCT4; for the experiment with the ΔN variant of OCT4, a magnification of ×92,000 was used. The pixel size was 1.94 Å (73,000) to 1.54 Å (92,000) per pixel on the object scale. The images were later analysed using the ImageJ software after matching the scale from the EM images.
Negative-stain image analysis
Several particles were picked using RELION (n = 450 for arrays in 3 mM MgCl2, n = 262 for arrays in 3 mM MgCl2 with OCT4 and n = 307 for arrays in 3 mM MgCl2 with the ΔN variant of OCT4). For particle picking, the images from the microscope were binned twofold in RELION and saved as 400 pixel × 400 pixel tiff files, which were later analysed using the ImageJ software54. First, the particles were encircled using the free-form selection tool in ImageJ. Later, the ‘set scale’ tool in ImageJ was used to set the size of the pixel in the image to 0.4 nm (pixel size of 0.2 nm at ×73,000 magnification multiplied by 2 for binning in RELION). The particle sizes were measured using the image analyser option in ImageJ and plotted.
Cryo-EM grid preparation and data collection
For cryo-EM of the OCT4-bound LIN28B nucleosome structure, we assembled an OCT4–SOX2–nucleosome complex as described. The sample was concentrated to 0.25 mg ml−1 for the cryo-EM grid. To avoid the extensive aggregation of the complex sample on the cryo-EM grid, OCT4 and SOX2 were mixed with nucleosomes in a 0.5:1 ratio during the assembly. The OCT4-bound nMATN1 nucleosome was assembled as described with a 2:1 ratio of OCT4 to nucleosome. Of the complex sample, 3 μl was applied to a freshly glow-discharged Quantifoil R2/1 holey carbon grid. The humidity in the chamber was kept at 95% and the temperature at +10 °C. After 5 s of blotting time, grids were plunge-frozen in liquid ethane using a FEI Vitrobot automatic plunge freezer.
For the LIN28B nucleosome and the OCT4-bound LIN28B nucleosome, electron micrographs were recorded on FEI Titan Krios at 300 kV with a Gatan Summit K3 electron detector using SerialEM55 (approximately 6,000 and approximately 11,000 micrographs, respectively) at the Cryo-EM facility at St. Jude Childrens’s Research Hospital. Image pixel size was 1.06 Å per pixel on the object scale. Data were collected in a defocus range of 7,000–30,000 Å with a total exposure of 90 e− Å−2. Fifty frames were collected and aligned with the MotionCorr2 software using a dose filter56,57. The contrast transfer function parameters were determined using CTFFIND4 (ref. 58). For the OCT4-bound nMATN1 nucleosome, the data were recorded on the FEI Titan Krios at 300 kV with a Falcon 4 electron detector using EPU (approximately 35,000 micrographs) at the Cryo-EM facility at the Dubochet Center for Imaging (DCI) at EPFL and UNIL. Data were collected in a defocus range of 7,000–25,000 Å. Image pixel size was 0.83 Å per pixel on the object scale.
Several thousand particles were manually picked and used for training and automatic particle picking in Cryolo59. Particles were windowed and 2D class averages were generated with the RELION software package60. Inconsistent class averages were removed from further data analysis. The initial reference was filtered to 40 Å in RELION. C1 symmetry was applied during refinements for all classes. Particles were split into two datasets and refined independently, and the resolution was determined using the 0.143 cut-off (RELION auto-refine option). All maps were filtered to resolution using RELION with a B-factor determined by RELION.
Initial 3D refinement was done with 2,600,000 particles. To improve the resolution of this flexible assembly, we used focused classification followed by focused local search refinements. Nucleosomes were refined to 2.8 Å. Density modification in Phenix improved the map to 2.5 Å (ref. 61). OCT4 bound to DNA (30 kDa) was refined to 4.2 Å using a subset of 65,000 particles after extensive sorting. Using density modification in Phenix, we improved resolution and the appearance of this density to 3.9 Å. The maps have extensive overlapping densities that we used to assemble the composite map and model. The LIN28B nucleosome sample contained 1,000,000 particles, which were refined to 3.1 Å, and improved with density modification to 2.8 Å.
For the second dataset, we collected 1,400 images, yielding 68,000 nucleosomal particles, which refined to 3.7 Å. Classification revealed that approximately 21,000 particles had OCT4 bound, which refined to 4.2 Å.
Molecular models were built using Coot62. The model of the nucleosome (Protein Data Bank (PDB): 6WZ5)63 was refined into the cryo-EM map in PHENIX64. The model of the OCT4 bound to DNA (PDB: 3L1P)19 were rigid-body placed using PHENIX, manually adjusted and rebuilt in Coot and refined in Phenix. Visualization of all cryo-EM maps was done with Chimera65.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.