• Download and Installation
• Data Flow and Design
• Creating a Simulated Target Map
• Preliminary Rigid-Body Registration
• Vector Quantization of the High-Resolution Structure with qpdb
• Vector Quantization of the Low-Resolution Map with qvol
• Flexible Docking with Molecular Dynamics
• Visualization
• Part II: Skeleton-Based Docking

Follow first the simple registration and download steps .

In addition to the executables, the Situs_2.3_flex_tutorial/bin directory contains 11 data files:

• 0_actin.str: X-PLOR stream file for setting up parameters and topology.
• 0_actin_orig.pdb: Original actin (start) structure.
• 0_actin_target.pdb: Target structure for flexible docking.
• 0_flex_target.inp: X-PLOR script for flexible docking using four codebook vectors.
• 0_toph19x.pro: Standard X-PLOR topology file for proteins.
• 0_toph19.pep: Standard X-PLOR peptide link.
• 0_param19.pro: Standard X-PLOR parameter file for proteins.

• 0_flex_rnap.inp: X-PLOR script for skeleton-based flexible docking using 15 codebook vectors.
• 0_rnap1.pdb: atomic structure of RNA polymerase in "closed" conformation.
• 0_rnap2.situs: simulated EM map of RNA polymerase in "open" conformation.
• 0_rnap.psf: X_PLOR psf file for setting up parameters and topology.

In the following, we will use the first 7 files to dock actin to various low-resolution maps. The user can compare all generated files to the files in the "solutions" directory. The other 4 files (brown) are used in part II of this tutorial.

Schematic diagram of flexing related routines. Major Situs components (blue) are classified by their functionality. The main data flow is indicated by brown arrows. The modeling of distance constraints for the motion capture skeleton is shown in dark blue. The external programs are shown in orange. Visualization (orange) for the rendering of the data requires a molecular graphics viewer (we recommend the free VMD graphics program, version 1.8.4 or higher; Chimera and Sculptor also support Situs format). The MD refinement is supported by X-PLOR.

Standard EM formats are supported and are converted to cubic lattices in Situs format. Subsequently, the data is inspected and, if necessary, prepared for the vector quantization using a variety of visualization and analysis tools. Atomic coordinates in PDB format can be transformed to low-resolution maps, if necessary, and vice versa. During vector quantization of the high-resolution structure, distances can be learnt that are sent to the vector quantizer of the low-resolution structure to enable skeleton-based fitting. After the vector quantization, the high-resolution structure is flexibly docked to the low-resolution density by the corresponding codebook vectors.

First we create a simulated EM map from a known atomic structure for later validation. We lower the resolution of the target structure to 15 Å with the pdb2vol kernel convolution utility. Enter at the shell prompt:

The program projects the atomic structure to the lattice, computes the Gaussian kernel, and carries out the real-space convolution, writing the resulting volumetric map to the file 1_actin_target.situs.

Before we start fitting the original actin structure to the target structure, it is mandatory to roughly align the atomic structure and the target map by rigid-body fitting.

Alternatively, an automated rigid-body fitting procedure can also be employed. E.g. the qrange tool has already been explained in the classic EM tutorial. However, if the conformational changes are significant, the best-scoring fit returned by rigid body fitting may not be the one we are interested in. It is therefore a good idea to export a number of best-scoring fits, and to explore the alignment of these fits by eye, before selecting one for subsequent flexible fitting.

Now, we perform the vector quantization of the roughly fitted structure with the qpdb utility.

and select mass-weighting (enter 2), ignore the B-factor cutoff (enter 1). Next, enter the number of codebook vectors: 4 (one for each of actin's subdomains). Watch the program compute a number of datasets (default: 8) for statistical averaging. The file 2_actin_orig_dock_target.qpdb now contains the four new codebook vectors, their rms variability, and the effective radius of their Voronoi cells, in PDB format. Finally, the user is asked whether nearest-neighbor connectivities should be learnt, or whether the Voronoi cells should be saved. Here we enter 1 (don't save the connectivities), but we want to save the Voronoi cells for flexible docking (enter 2). Enter the Voronoi cell filename: 2_actin_orig_dock_target.cell. This PDB-formatted file will now contain indices in each atom B-factor field denoting its membership to one of the four Voronoi cells.

The vector quantization of a volumetric datasets with the qvol utility involves only a few simple steps. After entering

the user is prompted to enter the density cutoff value. Again, we enter 10. Subsequently, we enter the number of codebook vectors, which must be compatible with the number for the high-resolution dataset (4). Finally, the user is asked whether nearest-neighbor connectivities should be learnt. This functionality is important later, but not now, so we enter no. The file 3_actin_target.qvol now contains the "QVOL" codebook vectors.

The most time-consuming part in setting up a simulation is the preparation of the input PDB and script files to conform with the topology and parameters provided by standard packages such as X-PLOR. This preparation usually requires considerable user experience, patience and a number of short test simulations. The necessary steps have already been implemented for actin. For details users should consult the X-PLOR documentation.

All stereochemical information and the system topology and parameters are defined in the stream file 0_actin.str in X-PLOR scripting language. This file will be sourced from all subsequent X-PLOR flexing scripts. It lists the required standard topology and parameter files (system dependent), and the amino acide sequence. The peptide links are formed here with the toph19.pep macro. Three standard atoms are renamed to conform with the input PDB files.

The flexible docking is enforced during an energy minimization with X-PLOR using NOE distance restraints that superimpose the centroids of the high-resolution Voronoi cells with the corresponding low-resolution vectors. Matching pairs of codebook vectors can be used as point landmarks for the flexible registration of the atomic structure with the target density map. The script file 0_flex_target.inp contains all the necessary commands. This file must be edited to assign pairs of corresponding vectors (the vector order generated by qvol and qpdb above is random). It is best to inspect the codebook vector files 3_actin_target.qvol and 2_actin_orig_dock_target.qpdb with a graphics program to assign the matching vectors. The order has already been assigned in 0_flex_target.inp, but the earlier calculations are machine-dependent, so a user will need to verify or adjust the assignment.

The X-PLOR script 0_flex_target.inp has several parts that are documented in the code. After reading the stream file 0_actin.str, topology and parameter settings are introduced for a dummy (probe) atom and for the codebook vectors (should not be altered by user). A segment statement creates the specified number of QVOL codebook vectors (adjust if necessary), and the Voronoi cells and atomic coordinates are read from a file. Subsequently, the QVOL vector coordinates are read. Finally, the NOE restraints are set, the QVOL vectors are matched with the Voronoi cells, and the system is energy minimized.

The result should look like this:

We are now prepared to improve the stereochemical quality of the flexing. It is possible to constrain the distances between the landmarks to reduce the effect of noise and experimental limitations on the codebook vector positions. This "skeleton" based approach, as described on the Vector Quantization page, is related to 3D motion capture technology used in the entertainment industry and in biomechanics. The application is demonstrated in the Flexible Docking Tutorial, Part II.