Name: Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination (Video) | JoVE Journal
Uploaded: 2022-08-17T13:30:00.000+00:00
Duration: 7 min 59 s
Description: Learn optimized nucleosome core particle preparation for cryo-EM structural studies using standard laboratory equipment. This method enables researchers to investigate DNA repair factor interactions with chromatin.

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5 minutes

We thank Dr. Mario Borgnia from the cryo-EM core at the National Institute of Environmental Health Sciences and Dr. Joshua Strauss from the University of North Carolina at Chapel Hill for their mentorship and training in the cryo-EM grid preparation. We also thank Dr. Juliana Mello Da Fonseca Rezende for technical assistance in the initial stages of this project. We appreciate the key contribution and support of the late Dr. Samuel H. Wilson and his lab members, especially Dr. Rajendra Prasad and Dr. Joonas Jamsen for the purification of the genetically fused APE1-Pol&#946; complex. Research has been supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences [grant numbers Z01ES050158, Z01ES050159, and K99ES031662-01].

1. Assemble nucleosome core particles via salt-gardient dialysis
NOTE: The preparation of nucleosome core particles using recombinant histone proteins for structural studies has been extensively described in detail by others14,15,16. Follow the purification of recombinant X. laevis histones and histone octamer assembly described by others14,15, and assemble the nucleosomal substrate as described below.
<ol>
	<li>Purchase three oligonucleotides (listed in Table 1) at the indicated scale: UND-197mer, 161mer, 35mer.
	<ol>
		<li>PAGE purify ultramers (197mer and 161mer) as described17.
		<ol>
			<li>Anneal equimolar amounts of oligonucleotides in 1x annealing buffer (10 mM Tris-Cl, pH 8.0, 50 mM NaCl). For example, mix 40 &#181;L of 161-mer [166.7 &#181;M]; 8 &#181;L of 35-mer [292.4 &#181;M]; 16.8 &#181;L of 197-mer comp strand [408.2 &#181;M]; 10 &#181;L of 10x anneal buffer and 10.9 &#181;L of dH2O. 
			NOTE: It is critical to control the annealing reaction with a temperature gradient. See Table 2 for the details on the temperature gradient.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform small-scale reconstitutions (scale by a factor of 1/57 of the example shown below for a final volume of 50 &#181;L) to determine the ratio of DNA to histone octamer (typical starting ratios of DNA:octamer are as follows: 1:0.9, 1:1.1, 1:1.2, 1:2; shown in Figure 2A); it is critical to determine this ratio empirically with small-scale reconstitutions.
	<ol>
		<li>After this ratio is determined, prepare a large-scale reconstitution. For example, mix 32.9 &#181;L of DNA-197 bp [99.4 &#181;M]; 1647.1 &#181;L of TE (1x); 1120 &#181;L of 5 M NaCl, and 62.3 &#181;L of WT histone octamer [2.56 &#181;M].</li>
	</ol>
	</li>
	<li>Make dialysis buffers: RBlow and RBhigh as described in Table 3 and Table 4, respectively; set up the reconstitutions as previously described14.
	<ol>
		<li>Transfer the dialysis tube into the beaker containing RBhigh, and allow for dialysis to proceed, with gentle stirring, for 16 h or until 2 L RBlow has been transferred into the waste beaker.</li>
	</ol>
	</li>
	<li>Transfer the dialysis tube into a 1 L beaker containing RB50mM buffer (Table 5) and allow the sample to dialyze for at least 3 h or overnight.
	<ol>
		<li>Evaluate reconstitution efficiency on a 6% polyacrylamide nondenaturing gel, ensuring there is less than 10% free DNA and a single NCP band; store NCPs at 4 &#176;C. See Figure 2A (lane 5) and Figure 2B for representative optimal reconstitution results.</li>
	</ol>
	</li>
</ol>
2. Prepare NCP-DNA repair factor complex (NCP-DRF)
<ol>
	<li>Purification by preparative-gel electrophoresis 
	NOTE: This method is the most labor-intensive (of the two described), and while it is effective at removing high molecular weight (HMW) species (Figure 6A) because of the high dilution factor, it can lead to some disassembly as shown in Figure 7A (NCP prep cell out lane), releasing DNA. However, up to 25% free DNA is still compatible with cryo-EM studies. Because of the high dilution, use this method to purify the NCP only, rather than the whole complex.
	<ol>
		<li>Prepare 70 mL of 6% nondenaturing polyacrylamide gel solution in 0.2x TBE, using polyacrylamide gel solution 37.5:1, acrylamide:bisacrylamide. Pour a cylindrical gel with outer radius of 28 mm and polymerize overnight.</li>
		<li>Assemble the preparative gel running apparatus, using a dialysis membrane with a 6-8 kDa MW cutoff. Use 0.25x TBE as the running buffer and 1x elution buffer (50 mM KCl, 10 mM HEPES, pH 7.5). Pre-run the cylindrical gel at a constant 12 W for 1 h, and collect fractions running the peristaltic pump at a flow rate of approximately 1 mL/min. 
		NOTE: If a UV detector is not available to identify fractions containing DNA, a screening experiment with 32P-NCP can be performed to identify the fractions of interest. Keeping all conditions the same, unlabeled NCPs can then be purified without a UV detector.</li>
		<li>Concentrate the 2.8 mL of NCP to 250 &#181;L using a centrifugal filter (MW cutoff 30 kDa), and load onto the preparative gel and electrophorese for a total of 6 h. At 2 h, when the xylene cyanol has ran out of the gel, start collecting 1.5 mL fractions. 
		NOTE: At 2.5 h, fractions will be clear of xylene cyanol; the DNA (197mer) elutes at approximately 3-3.5 h, and the NCP is expected to elute at 4.5-5 h mark.</li>
		<li>Analyze fractions of interest on a 6% nondenaturing polyacrylamide gel. Pool fractions containing the NCP, and immediately concentrate with a centrifugal filter (MW cutoff 30 kDa) to 1 mg/mL. The next day, evaluate the quality of the NCP on a 6% polyacrylamide nondenaturing gel before complexing with the DNA repair factor (DRF) of interest.</li>
		<li>Incubate the NCP and the DRF of interest on ice for 15 min using an optimal buffer for the specific complex. For example, mix 1,000 &#181;L of NCP [1.2 &#181;M]; 260 &#181;L of 5x binding buffer (250 mM HEPES, pH 8, 500 mM KCl, 25 mM MgCl2), 22 &#181;L of 3 M KCl, and 18 &#181;L of MBP- Pol &#946;-APE1 [338 &#181;M]. 
		NOTE: The temperature and ionic strength at which the complex is made may need optimization&#160;for different complexes.</li>
		<li>Add glutaraldehyde (freshly opened; EM grade) to a final concentration of 0.005%. Therefore, to this reaction, add 26.8 &#181;L&#160;of 0.25% glutaraldehyde with 13.2 &#181;L of dH2O. Mix well, and incubate at room temperature for 13 min.</li>
		<li>Quench with 1 M Tris-Cl, pH 7.5, to a final concentration of 20 mM Tris-Cl, pH 7.5. Concentrate to ~50 &#181;L and exchange the buffer with 1x freezing buffer: 50 mM KCl, 10 mM HEPES, pH 7.5 using a desalting column. 
		CAUTION: Glutaraldehyde can cause severe skin burns and eye damage; it is harmful if swallowed, and it is toxic if inhaled. Wear a lab coat, goggles, gloves, and handle glutaraldehyde in a fume hood and&#160;wear a mask. 
		NOTE: It is critical to perform the crosslinking reaction in a large volume with the NCP at this lower concentration. Previous attempts of crosslinking, even at this concentration of glutaraldehyde, but at a 10x higher concentrated NCP led to aggregation of the sample and over-crosslinking as indicated by SDS PAGE and size exclusion chromatography. These grids had no discernable particles with just clumps (Figure 5D,E).</li>
		<li>Determine the absorbances at OD280 and OD260 and concentrate further if needed to reach 1.3-3 mg/mL, based on OD280 (typical ratio of OD260/ OD280= 1.7-2 yields good particles). For this small volume of 50 &#181;L, the best method to reduce sample loss is to make a dialysis button with a lid of a 1.7 mL tube covered by a dialysis membrane (6-8 kDa MW cutoff), cutting the bottom of the tube and using the rim of the tube to seal the membrane on top of the lid.</li>
		<li>Place the dialysis button containing the sample on top of a polyethylene glycol bed, with the membrane facing down (check progress every 2 min). This method is preferred where the concentration needed is less than or equal to 2.5-fold to avoid diluting or losing the sample in the concentrator. It is critical to immediately prepare cryo-EM grids of the complex after this step.</li>
	</ol>
	</li>
	<li>Purification by size exclusion chromatography
	<ol>
		<li>The day before freezing, wash and equilibrate a size exclusion column with 60 mL of dH2O, followed by 80 mL of freezing buffer (50 mM KCl, 10 mM HEPES, pH 8) overnight at a rate of 0.4 mL/min. Using NCPs straight after reconstitution, prepare the same complex using 2.5x greater amounts as follows: mix 2,500 &#181;L of NCP [1.2 &#181;M]; 650 &#181;L of 5x binding buffer; 45 &#181;L of MBP-Pol &#946;-APE1 [338 &#181;M] and 55 &#181;L of 3M KCl.</li>
		<li>Incubate the mixture on ice without crosslinker for 15 min. Then, add glutaraldehyde to a final concentration of 0.005% as described in the preparative gel method. In this case, however, concentrate the complex in a pre-equilibrated (with freezing buffer) centrifugal filter to approximately 120 &#181;L.</li>
		<li>Immediately analyze the peak fractions and concentrate those fractions containing the histones and MBP-Pol &#946;-APE1 as indicated previously. See Figure 5A,B,C for successful results using the size exclusion method&#160;and Figure 7&#160;using both methods.</li>
	</ol>
	</li>
</ol>
3. Freeze nucleosomal complex
<ol>
	<li>After turning on the plunge freezer and filling the humidifier with 50 mL of dH2O, set the chamber temperature to 22 &#176;C and HR (humidity) to 98%. Place the ethane cup and liquid nitrogen cup (containing a labeled grid box) in their respective space holders.</li>
	<li>Cover the ethane cup with the ethane lid dispenser, and carefully pour liquid nitrogen (LN2) over it, while also filling the LN2 cup with LN2. When the LN2 level has stabilized and reaches 100%&#160;and a temperature of -180 &#176;C, carefully open the ethane valve and fill the ethane cup until it forms a bubble on the clear lid. Remove the ethane dispenser.</li>
	<li>Place two filter papers onto the blotting device and secure them with a metal ring. Go to the set up and use the following parameters for blotting: 0 pre-blot, 3 s blot, 0 post-blot; select A-plunge and click on OK.</li>
	<li>On the main screen, click on Load Forceps, and load them with a grid with the carbon/application side facing toward the left (prepared as before). Calibrate the forceps adjusting the Z-axis to ensure a solid blot. Click on Lower Chamber and apply 3 &#181;L of the complex (1.3-3 mg/mL; OD280). Click on Blot/A-plunge. This will rotate the grid to blot from the front and will plunge freeze it.</li>
	<li>Transfer and store the grid in the grid box in the LN2 chamber. When all four grids have been frozen and placed in the grid box, rotate the lid to neutral position, where all the grids are covered by the lid, and tighten the screw. Grids can be stored in LN2 until screening is initiated.</li>
</ol>
4. Screen grids
<ol>
	<li>Before loading the samples in the microscope, place the vitrified grids on a ring and secure them using a C-clip. Perform this clipping process under LN2 in a humidity-controlled room, to avoid ice contamination.</li>
	<li>When loading the microscope, insert the grids in a 12-slot cassette. Shuttle the cassette into a nanocab capsule and load it into the autoloader. The autoloader robotic mechanism initiates the process.</li>
	<li>Transfer the grid from the cassette to the microscope stage. Adjust the stage to eucentric height by wobbling the stage 10&#176; and simultaneously moving the Z-height until minimal planar shift is observed in the images.</li>
	<li>Once the eucentric height is reached, start imaging. First, obtain the atlas by taking a 3 x 3 montage image of the grid, in which each montage is taken at 62x magnification. Pick three squares of varying sizes; take the eucentric height, and then image at 210x magnification.</li>
	<li>Once a square is imaged, pick one hole each from the edge, center, and in between within the squares. Image each hole at 2600x magnification.</li>
	<li>Before taking this high magnification image, the autofocusing occurs at an offset of the imaging area. Take the high magnification image from the center of the hole at 36,000x magnification and at 7.1 s exposure time, 60 frames, 1.18-pixel size, and 3 &#181;m defocus. See representative images of screening in Figure 5, Figure 6, and&#160;Figure 7.</li>
</ol>

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<li>Rodriguez, Y., Smerdon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structural+location+of+DNA+lesions+in+nucleosome+core+particles+determines+accessibility+by+base+excision+repair+enzymes%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">The structural location of DNA lesions in nucleosome core particles determines accessibility by base excision repair enzymes.</a> The Journal of Biological Chemistry. 288 (19), 13863-13875 (2013).</li>
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</ol>

The authors declare no competing interests.

A specific protocol for purifying the DNA repair factor will be dependent on the enzyme of interest. However, there are some general recommendations, including the use of recombinant methods for protein expression and purification18; if the protein of interest is too small (&#60;50 kDa), structure determination by cryo-EM had been nearly impossible until more recently through the use of fusion systems19, nanobody-binding scaffolds20, and optimizing i...

Eukaryotic DNA is organized and compacted by histone proteins, forming chromatin. The nucleosome core particle (NCP) constitutes the fundamental repeating unit of chromatin that regulates accessibility to DNA-binding proteins for DNA repair, transcription, and replication1. Although the first X-ray crystal structure of the NCP was first solved more than two decades ago2 and many more structures of the NCP have been published since3,4,5,6, DNA repair mechanisms in nucleosomal substrates have not yet been delineated. Uncovering the molecular details underlying DNA repair in chromatin will require structural characterization of the participating components to understand how local structural features of the NCP regulate DNA repair activities. This is particularly important in the context of base excision repair (BER) given that biochemical studies with BER enzymes suggest unique DNA repair mechanisms in nucleosomes that are dependent on enzyme-specific structural requirements for catalysis and the structural position of the DNA lesion within the nucleosome7,8,9,10,11,12,13. Given that BER is a vital DNA repair process, there is considerable interest to fill in these gaps while also establishing a starting point from which other technically feasible structural studies involving relevant nucleosomal complexes can be carried out.
Cryo-EM is rapidly becoming the method of choice for solving the three-dimensional (3D) structure of complexes whose large-scale preparation of homogeneous sample is challenging. Although, the design and purification of NCPs complexed with a DNA repair factor (NCP-DRF) will likely necessitate tailored optimization, the procedure presented here to generate and freeze a stable NCP-DRF complex provides details on how to optimize the sample and cryo-EM grid preparation. Two workflows (not mutually exclusive) shown in Figure 1, and the specific details in the protocol identify critical steps and provide strategies for optimizing these steps. This work will propel the chromatin and DNA repair field in a direction where complementing biochemical with structural studies becomes technically feasible to better understand the molecular mechanisms of nucleosomal DNA repair.

<table><tbody><tr><td>1 M HEPES; pH 7.5</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>1 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Thermo Fisher Scientific</td><td>AM9530G</td><td></td></tr><tr><td>10x TBE</td><td>Bio-rad</td><td>1610733</td><td></td></tr><tr><td>25% glutaraldehyde</td><td>Fisher Scientific</td><td>50-262-23</td><td></td></tr><tr><td>3 M KCl</td><td>Thermo Fisher Scientific</td><td>043398.K2</td><td></td></tr><tr><td>491 prep cell</td><td>Bio-rad</td><td>1702926</td><td></td></tr><tr><td>Amicon Ultra 15 centrifugal filter (MW cutoff 30 kDa)</td><td>Millipore Sigma</td><td>Z717185</td><td></td></tr><tr><td>Amicon Ultra 4 centrifugal filter (MW cutoff 30 kDa)</td><td>Millipore Sigma</td><td>UFC8030</td><td></td></tr><tr><td>AutoGrid Tweezers</td><td>Ted Pella</td><td>47000-600</td><td></td></tr><tr><td>Automatic Plunge Freezer</td><td>Leica</td><td>Leica EM GP</td><td></td></tr><tr><td>C-1000 touch thermocycler</td><td>Bio-rad</td><td>1851148</td><td></td></tr><tr><td>C-clips and rings</td><td>Thermo Fisher</td><td>6640--6640</td><td></td></tr><tr><td>Clipping station</td><td>SubAngostrom</td><td>SCT08</td><td></td></tr><tr><td>Dialysis Membrane (MW cufoff 6-8 kDa)</td><td>Fisher Scientific</td><td>15370752</td><td></td></tr><tr><td>Diamond Tweezers</td><td>Techni-Pro</td><td>758TW0010</td><td></td></tr><tr><td>dsDNA</td><td>Integrated DNA techonologies</td><td>N/A</td><td></td></tr><tr><td>FEI Titan Krios</td><td>Thermo Fisher</td><td>KRIOSG4TEM</td><td></td></tr><tr><td>FPLC purification system</td><td>AKTA Pure</td><td>29018224</td><td></td></tr><tr><td>Fraction collector Model 2110</td><td>Bio-rad</td><td>7318122</td><td></td></tr><tr><td>Glow Discharge Cleaning System</td><td>Ted Pella</td><td>91000S</td><td></td></tr><tr><td>Grid Boxes</td><td>SubAngostrom</td><td>PB-E</td><td></td></tr><tr><td>Grid Storage Accessory Pack</td><td>SubAngostrom</td><td>GSAX</td><td></td></tr><tr><td>Liquid Ethane</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Minipuls 3 peristaltic two-head pump</td><td>Gilson</td><td>F155008</td><td></td></tr><tr><td>Nanodrop</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Novex 16%, Tricine, 1.0 mm, Mini Protein Gels</td><td>Thermo Fisher Scientific</td><td>EC6695BOX</td><td></td></tr><tr><td>Pipetman</td><td>Gilson</td><td>FA10002M</td><td></td></tr><tr><td>Pipette tips (VWR) Low Retention</td><td>VWR</td><td>76322-528</td><td></td></tr><tr><td>Polyacrylamide gel solution (37.5:1)</td><td>Bio-rad</td><td>1610158</td><td></td></tr><tr><td>polyethylene glycol (PEG)</td><td>Millipore Sigma</td><td>P4338-500G</td><td></td></tr><tr><td>Pur-A-lyzer Maxi 3500</td><td>Millipore Sigma</td><td>PURX35050</td><td></td></tr><tr><td>Purified recombinant DNA repair factor</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>R 1.2/1.3 Cu 300 mesh Grids</td><td>Quantifoil</td><td>N1-C14nCu30-01</td><td></td></tr><tr><td>Recombinant histone octamer</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Spring clipping tools</td><td>SubAngostrom</td><td>CSA-01</td><td></td></tr><tr><td>Superdex 200 column 10/300</td><td>Millipore Sigma</td><td>GE28-9909-44</td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>Thermo Fisher</td><td>Talos Arctica 200 kV</td><td></td></tr><tr><td>Tweezers Assembly for FEI Vitrobot Mark IV-I</td><td>Ted Pella</td><td>47000-500</td><td></td></tr><tr><td>UltraPure Glycerol</td><td>Thermo Fisher Scientific</td><td>15514011</td><td></td></tr><tr><td>Vitrobot</td><td>Thermo Fisher</td><td>Mark IV System</td><td></td></tr><tr><td>Whatman Filter paper (55 mM)</td><td>Cytiva</td><td>1005-055</td><td></td></tr><tr><td>Xylene cyanol</td><td>Thermo Fisher Scientific</td><td>440700500</td><td></td></tr><tr><td>Zeba Micro Spin Desalting Columns, 7K MWCO, 75 &amp;micro;L</td><td>Thermo Fisher Scientific</td><td>89877</td><td></td></tr></tbody></table>

preparation of nucleosome core particles complexed with dna repair factors for cryo-electron microscopy structural determination

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Properly assembled NCPs (Figure 2) were used to make a complex with a recombinant fusion protein of MBP-Polβ-APE1 (Figure 3). To determine the ratio of NCP to MBP-Polβ-APE1 to form a stable complex, we performed electrophoretic mobility shift assays (EMSA) (Figure 4), which showed a singly shifted band of the NCP with 5-fold molar excess of MBP-Polβ-APE1. During the optimization of making this complex, crosslinking with glutaraldehy...

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

preparation-nucleosome-core-particles-complexed-with-dna-repair

Research

JoVE Journal

Biochemistry

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA.  We also describe how sedimentation velocity experiments in the analytical ultracentrifuge,  and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Single Particle Cryo-Electron Microscopy: From Sample to Structure

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Optimizing Sample Preparation for Cryogenic Electron Microscopy

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Learn optimized nucleosome core particle preparation for cryo-EM structural studies using standard laboratory equipment. This method enables researchers to investigate DNA repair factor interactions with chromatin.

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination (Video) | JoVE Journal

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

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