FokI cleavage complexes modeled with both parallel (left) and anti-parallel (right) recognition sites. The models were generated based on the crystal structures of the FokI-20-mer10 and the BamHI-DNA complexes15. The DNAs were extended. Both recognition domains (RD1 and RD2) are bound to their respective recognition sites and the cleavage domains (CD1 and CD2) form a BamHI-like dimer on DNA1. The arrows indicate the direction of the recognition sequence in each DNA. The parallel model provides for protein-protein and possibly protein-DNA interactions, not seen in the anti-parallel model, that could further stabilize the cleavage complex. (Reproduced by permission from Vanamee et al.13)

2D projection matches generated a) from model 1 and b) from model 2. The left panels show representative projections generated from the models at 9° intervals and the right panels show the matched classified particles. The numbers in the right panels indicate the number of particles in each projection. The atomic models used to generate the 2D projections are also shown on the sides respectively. Model 2 is crossed out indicating that it is not supported by the EM data. The coordinates of the DNAs were omitted as DNA positively stains with uranyl acetate and cannot be visualized in these experiments. The program EMAN (v.1.7)17 was used for particle selection and data analysis. A total of 2,316 initial particles were selected with a window size of 104 pixels.

Close-up view of a raw CCD image showing uniform particles of a) the FokI/DNA complex and b) the FokI monomer. a) FokI was mixed with an excess of double stranded oligonucleotide (1:1.2) to ensure that all protein was in complex with DNA. The mixture was diluted to a final concentration of ~3 μM for the electron microscopic studies. 4 μl of such prepared complex was added to carbon-coated 400 mesh square Cu grids. The samples were negatively stained by 2% uranyl acetate. Excess solution was blotted away, and the grids were air-dried prior to loading them into the microscope. Data were collected on a Tecnai F20 electron microscope equipped with a field emission gun (FEG) and operated at 200 keV in low-dose mode. Specimens were imaged at room temperature in a Gatan specimen holder. Dozens of images were taken with a 4k × 4k charge-coupled device (CCD) camera (F415 from Tietz Video and Imaging Processing GmbH, Gauting, Germany) at 50,000 × magnification. b) the FokI monomer was prepared and imaged at identical conditions but in the absence of DNA. c) Reference-free class averages of the FokI/DNA complex on top and the corresponding high-resolution surface models below. In the models, the second recognition domain (RD2) is colored in red. The three class averages from left to right were created from 108, 86, and 75 particles respectively.