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Amicon Ultra-0.5 ml centrifugal filters (Millipore-Sigma, UFC503008, pore size: 30 kDa NMWCO)
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Nonwoven cleanroom wipes: TX604 TechniCloth (TexWipe, catalog number: TX604)
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Petri dish (Fisher Scientific, catalog number: 08-757-100A)
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Standard disposal cuvette (Perfector Scientific, catalog number: 9002)
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Distilled deionized H2O (DDI H2O)
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pUC19 Vector (New England Biolabs, catalog number: N3041S)
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PCR primers (IDT, custom order)
F364: 5’-GAGTTCTTGAAGTGGTGGCC-3’
R364: 5’-GGTAACTGTCAGACCAAGTTTACTC-3’
F480: 5’-GCGATTAAGTTGGGTAAC-3’
R480: 5’-GTTCTTTCCTGCGTTATC-3’
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DreamTaq polymerase (Thermo Fisher Scientific, catalog number: EP0701)
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Deoxynucleotide (dNTP) Solution Mix (New England Biolabs, catalog number: N0447S)
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PCR purification kit (Qiagen, catalog number: 28104)
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Restriction endonuclease: DdeI (New England Biolabs, catalog number: R0175S)
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Restriction endonuclease: BspQI (New England Biolabs, catalog number: R0712S)
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CutSmart® Buffer (New England Biolabs, catalog number: B7204S)
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Oligonucleotide (IDT, custom order)
O30: 5’-TCATCTGCGTATTGGGCGCTCTTCCGCTTCCTATCT-3’
O31: 5’-TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCATA-3’
O32: 5’-GCTTATGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCTTGCGCAGCGAGTCAGTGAGATAGGAAGCGGAAGAGCGCCCAATACGCAGA-3’
O33: 5’-CACTGACTCGCTGCGCAAGGCTAACAGCATCACACACATTAACAATTCTAACATCTGGGTTTTCATTCTTTGGGTTTCACTTTCTCCAC-3’
O34: 5’-CTAACAGCATCACACACATTAACAATTCTAACATCTGGGTTTTCATTCTTTGGGTTTCACTTTCTCCACCACTGACTCGCTGCGCAAGG-3’
O36: 5’-TACGTGTAGGAATTATATTAAAGAGAAAGTGAAACCCAAAGAATGAAAAAGAAGATGTTAGAATTGTAAGCGGTATCAGCTCACTCATA-3’
O37: 5’-GCTTATGAGTGAGCTGATACCGC-3’
O42: 5’-TCATGACTCGCTGCGCAAGGCTAACAGCATCACACACATTAACAATTCTAACATCTGGG TTTTCATTCTTTGGGTTTCACTTTCTCCAC-3’
O43: 5’-CCTTGCGCAGCGAGTCA-3’
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T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201S)
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T4 DNA Ligase (Thermo Fisher Scientific, catalog number: 15224090)
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DL-Dithiothreitol (Sigma-Aldrich, catalog number: 43819-5G)
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EDTA (Thermo Fisher Scientific, catalog number: 15576028)
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Tris base (Sigma-Aldrich, catalog number: 10708976001)
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Phenol:Chloroform:Isoamyl Alcohol 25:24:1, Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA (Sigma-Aldrich, catalog number: P3803-100ML)
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Isopropanol (Fisher Scientific, catalog number: A426P-4)
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Ethanol (Decon Labs, catalog number: 2701)
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Sodium acetate buffer solution, pH 5.2±0.1 (25 °C), 3 M, 0.2 μm filtered (Sigma-Aldrich, catalog number: S7899-100ML)
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Acetic acid (ACROS Organics, catalog number: AC124040010)
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HCl (Sigma-Aldrich, catalog number: 258148-25ML)
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Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266-100G)
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Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-500G)
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Muscovite Block Mica (AshevilleMica, catalog number: Grade-1)
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1-(3-Aminopropyl) silatrane (APS) [synthesized as described in ref. (Shlyakhtenko et al., 2013)]
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TESPA-V2 AFM probe (Bruker AFM Probes, catalog number: TESPA-V2)
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Platinum coated calibration grid, 1 µm × 1 µm period (Bruker AFM Probes, catalog number: PG)
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10× binding buffer (see Recipes)
The AFM images were analyzed using the FemtoScan Online software package (Advanced Technologies Center, Moscow, Russia). Graphs were made by Origin software (OriginLab Corporation, Northampton, MA, USA).
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Measure the contour length. Open the flattened image in FemtoScan, measure and record the contour length of the free DNA from one end to the other. For the internal length calibration, use the measurements to generate a histogram and fit it with a normal (Gaussian) distribution. To obtain the calibration factor, divide the peak center (Xc) by the substrate length into base pairs.
Note: The calibration factor should be ~0.34.
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Measure the position of the protein (Figure 2). Start from the end of the shorter arm on the DNA substrates towards the center of the protein to obtain the position of the protein (arm length 1, shown in Figure 2). Keep recording from the center of the protein towards the other end of the DNA substrate to obtain the contour length of the DNA (arm length 1 and arm length 2 together, shown in Figure 2). Divide each arm length by the calculated calibration factor to obtain arm lengths in DNA base pairs. A histogram for the distribution of the protein can be made from the dataset of arm length 1 (Figure 2C).

Figure 2. Measurement of the protein position. A. Representative AFM image (0.5 × 0.5 µm) of F5 with PriA. The Z-scale is 3 nm. Arrow points to bound PriA on the F5 DNA substrate. B. Zoomed-in image (0.25 × 0.25 µm) with the dotted line showing the contour length measurement. The position of each protein is measured from the end of the shorter arm to the center of the protein (dotted green line), and the total length of the protein-bound DNA substrate was measured continuously from the center of the protein to the end of the other arm (dotted blue line). C. Histogram for PriA position on F5 DNA using the data of the short arm length. The histogram was fitted by Gaussian with a single peak centered at 254 ± 23 bp (S.D.), and with a bin size of 20 bp.
For the double-feature complexes in the SSB-PriA-DNA results, measure the height of each feature using the cross-section feature of the software as described below, and assign the taller one to SSB. For the length measurement, start from the end closer to the SSB, continuously measure and record towards the center of SSB and PriA, keep on recording until the end of the other side of the DNA substrate. Plot these values as a chart by setting the DNA length as clustered column and protein position as scatter (Figure 3).

Figure 3. Measurement of the protein distribution of the complexes containing PriA and SSB proteins. A. Representative AFM image of the PriA+SSB+fork DNA (0.5 × 0.5 µm). The Z-scale is 3 nm. B. The zoomed-in image (0.25 × 0.25 µm) of the double-protein complex with the dotted line showing the contour length measurement. The green arrow directs to PriA in the complex, while the SSB position is directed by the blue arrow. C. Map of the proteins on the F3 DNA substrate with the SSB position corresponding to zero value. Blue squares indicate the position of SSB and the red dots point to the PriA position.
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Measure the volume of the protein. For each protein, collect two sets of height (H) and full width at half maximum values (D) from orthogonal cross-section measurements (Figure 4A). Apply the measurements to the formula as described in Gilmore et al. (2009): V = 3.14 × H/6 × (0.75 x D1 x D2 + H2). Plot these values as a histogram and fit the peak(s) with a normal (Gaussian) distribution to obtain the volume of the protein population.
Note: In our AFM-imaging system (MultiMode 8, Nanoscope V system, E scanner), the accuracy of the vertical measurement is at the sub-nm level (Lyubchenko, 2011). The measured height of DNA in the air is usually around 0.6 to 0.8 nm as performed in our and other laboratories (Thomson et al., 1996; Hansma et al., 1996; Maeda et al., 1999; Pietrasanta et al., 1999; Ye et al., 2000; Kato et al., 2002; Shlyakhtenko et al., 2003), and it can be taken as a control for the height measurements of the protein-DNA complexes. Additionally, the measured thickness of the supported phospholipid bilayer is 4-5 nm (Lv et al., 2018), which is in perfect agreement with the bilayer measurements obtained by other methods.

Figure 4. Measurement of the size of the protein. A. Representative AFM image of the double-protein complex (0.2 × 0.2 µm). Cross-section profiles (green and blue lines) of a protein produce the height distribution curves shown in (B-E): (B) and (C) are the plots for SSB, and (D) and (E) show the height distributions for PriA. From these curves, height (H) and width (D) values are used for calculating the protein volume.
The work was supported by the National Institutes of Health grants R01 GM118006 to YLL and R01 GM100156 to PRB and YLL. This protocol was adapted from a previously described work (Wang et al., 2020).
There are no competing interests to declare.