The overall goal of this procedure is to bring the 3D NA suite of programs to laboratory scientists and others to investigate DNA and RNA spatial organization with state-of-the-art computational tools. This is accomplished by first downloading the software from the 3D NA forum and installing it on a local computer. The second step is to analyze a representative DNA structure at the level of the constituent base pair steps.
Next, the same DNA structure is modified at the base pair step level, and is then rebuilt using the newly defined base pair step parameters. The subsequent step is the analysis of a large ensemble of related structures. The final step is the alignment of a set of related DNA structures on a common reference frame in order to visualize global distortions of the structures.
Ultimately, the 3D NA tools are used to examine how individual base pair steps, including those bound to drugs and proteins, contribute to the overall folding of DNA and to identify the sites and directions of sizable global distortions. The main advantage of this technique over existing methods is that the software is robust and widely applicable to a variety of different nucleic acid containing structures. The method can help answer key questions in biochemistry and biophysics, such as how genomes are organized in three dimensions, and what kind of deformation DNA can adopt.
To begin installation of the software package, connect to the three DA website@xthreed. org and click the link to the 3D NA forum Within the forum. Select the register link and follow the instructions to create a new account.
Once a username and password have been established, log into the forum, select the downloads link in the welcome section, and on the new page, choose 3D NA download. Double click on the tar gz file to create a folder named X 3D NA dash V 2.1, which contains the 3D NA software. Open the terminal application, typically found under utilities on a Macintosh, and change to the X 3D NA dash V 2.1 directory.
Run the setup script needed for 3D NA to function properly. All commands used in this video can be found in the text protocol. Next, copy the two line export settings that appear printed on screen.
If the user has placed the software in the applications directory, the two lines should appear as shown here. Open the text edit application found in the applications directory. Under the format menu, select Make plain text.
If a BRC file exists, open the file in text, edit and paste the export settings at the end of the file. If no, bash RC file exists, paste the export settings into a blank file and save with the file name bash RC in the home directory. In order for the new settings to be available, run the newly saved Bash RC file within the terminal program.
This video demonstrates the analysis of a representative nucleic acid structure, the DNA bound to the HBB protein from the bacterium Borrelia bergdorf eye. The first step in the 3D NA analysis is the creation of a file that lists all of the paired bases. This is done by typing a command to apply the Find para program to the atomic structure stored in the file to NP two, MP two pdb.
The next step in the analysis is to determine the geometric parameters that characterize the structure. The command analyze takes as the input the base pair file and creates several output files which appear in the working directory. The geometric values can then be calculated by typing, analyze two MP two bps.
The generated output files include two MP two out, which contains an overview of the calculated parameters and bpco step par, which contains a list of the rigid body parameters in addition to the analysis of a nucleic acid structure, the 3D NA software provides the ability to build a structural model from rigid body parameters using the rebuild command in order to create an approximate atomic model, which includes coordinates for both backbone and base atoms. Type the command listed in the text. This command instructs 3D NA to introduce a standard BDNA backbone confirmation in the construction of models from rigid body parameters.
A double helical model with the rigid body parameters found in the H-B-B-D-N-A structure can then be built by typing the appropriate command. The bpco step par file can be edited to generate a modified structure. Here, the extreme roll angles formed at the two sites of greatest bending are modified.
The structure is then built with the modified set of step parameters as the input file. The two models can be viewed with a standard molecular viewer in contrast to the analysis of a single nucleic acid containing structure. This portion of the video will show how to analyze a large ensemble of structures in a set of files, starting with the names MD underscore set one PDB and ending with a number.
The first step in the analysis is identification of the paired bases obtained by running the find pair program as before, since the entire set of structures shares the same base pairing scheme, one needs to run find pair once on a representative file here, the file ending with 1001. This generates the base pairing information and stores it in the file MD at set one BPS PS.The next step finds the geometric parameters of the entire set of structures using the Command X 3D ncore ensemble.Analyze. Then the desired rigid body parameters can be extracted from the overview output file MD at set one out.
This is performed using the Command X three DCO ensemble extract. The roll angles are extracted and a comma separated text file of the roll angles is created. This file can be read into other programs for further analysis and plotting.
The most recent version of 3D NA has a new feature that allows a user to look at multiple structures from a common perspective. The X three DCO ensemble reorient command superposes a collection of related structures on a common base pair or base pair step, as in previous protocols. The first step is to calculate the base pairing of the DNA with the find pair program.
In this case, the input file is two KEK pdb the file containing the 10 structures of the CIA coli, O 3D NA operator bound to the headpiece of the LAC Repressor protein. The Command X three DCO ensemble reorient will align the individual models in a multi-model structure on a common reference frame. This command requires both the PDB file and the base pairing file from the previous step as input.
Also included in the command is the identity of the base pair step against which all of the structures are aligned. The W 3D NA web interface includes some popular features of the 3D NA software package. This portion of the video draws attention to the capability to construct three dimensional models of protein decorated DNA with the web server.
The first step in building a protein decorated DNA model is to visit the W 3D NA website located at W three d.rutgers.edu. Selection of reconstruction from the menu at the top left hand side of the page activates the online model building capabilities. The next step is to select the bound protein DNA template link found in the lightly shaded box in the middle of the new page.
This selection will activate a pulldown menu, which allows the user to specify the number of bound proteins. Selection of the number of bound proteins leads to a new specification page. The user can then type or paste in the desired sequence.
In the text box, in the middle of the new page, there is a pull down menu below the text box to select the helical confirmation of the unbound regions of DNA. There is a text box near the bottom of the specification page to enter the binding positions and file names of the bound proteins. The binding position specifies the location of the center of the protein bound fragment on the DNA sequence.
At the bottom of the specification page, there is a box that allows the user to generate a preview of the protein bound DNA. In this case, that box is checked and the continue button is clicked. This action generates a review page listing the selected parameters, as well as any errors that may have arisen from the overlap of the selected binding sites.
The user can select the back button if any changes need to be made, or the build button to proceed. The next page displays a static image of the DNA protein complex in its most extended arrangement and allows for online interactive visualization via and web. The user can also download a file containing atomic coordinates.
The 3D NA software tools are routinely used to analyze nucleic acid structures. The values of the rigid body parameters reveal distortions in three dimensional structure, such as the two sites of extreme DNA bending into the major groove with large positive roll angles found at at steps 13 and 22. In the crystal complex of DNA with the Borrelia Bergdorf, RHBB protein.
The capability of the software to rebuild structures from these quantities makes it possible to determine how individual base and base pair steps contribute to the overall molecular fold. As illustrated here, the global bending of DNA induced by HBB reflects more than the two extreme role distortions noted above. The DNA remains highly curved when reconstructed with these base pair steps straightened.
That is when the two largest values of the role have been set to zero. The new capability in 3D NA to examine large numbers of related structures makes it possible to extract both sequence and time dependent patterns in the spatial arrangements of simulated DNA and RNA molecules. For example, the yellow color coating of the roll angles between successive base pairs in two large sets of simulated DNA structures reveals the preferential bending of these molecules at Permian purine base pair steps.
The higher values of roll depicted in red that persist for short periods at the ends of the DNA are suggestive of localized melting and re kneeling of the double helic structure. The variational patterns of other rigid body parameters, such as the angles and distances between complementary bases can help to decipher the precise structural distortions. The capability of the 3D NA software to reorient related molecules in a common reference frame reveals features of overall structure hidden in many of the files stored in the protein data bank.
For example, the conventional alignment of related structures on the basis of a root mean square fit of corresponding atoms produces a series of similar spatial pathways that roughly superimpose upon one another here, the 10 NMR based models of the Esia coli, O 3D NA operator bound to the head pieces of the LAC repressor protein, the super position of the same structures on a common coordinate frame on the five prime terminal base pair of each duplex reveals sizable distortions of global structure in which the molecules flex in appreciably different directions. The structural variability may influence the ease with which the CIA coli LAC repressor protein binds O three and induces a loop between O three and sequentially distant operators in the LAC operation. As illustrated here, the precise placement of an architectural protein like HBB from Borrelia burgdorferi can have a dramatic effect on the overall folding of DNA.
If two copies of the known high resolution structure are separated by 43 base pairs, an 81 base pair, DNA fragment closes into a tight, nearly circular configuration. If the two proteins are separated by an additional five base pairs, the DNA follows an open meandering pathway. The very different arrangements of the protein decorated duplex show how the spacing of architectural proteins can affect the cyclization or looping of DNA After its development.
This technique paved the way for researchers in the field of nucleic acid structural biology to explore DNA protein interactions, RNA, folding and nanoparticle design in order to gain a better understanding of developmental defects and diseases such as cancer and design new therapeutics.
