• Download and Installation
• Data Flow and Design
• Preparing the Volume with Map Editing
• Preliminary Rigid-Body Registration
• Flexible Refinement with ddforge
• Residue Constraints
• Visualization
• Part II: Feature-Based Flexible Fitting

First, follow these registration and download steps (each Situs tutorial is separate and must be downloaded and compiled individually)!

Then, return to this page.

• 0_lactoferrin_orig.pdb: Start structure of lactoferrin basen on PDB entry 1lfg.
• 0_lactoferrin_target.situs: Target structure for flexible fitting (PDB entry 1lfh blurred to 7A).
• 0_residue_codes_flexible.txt: Residue codes that keep the structure flexible.
• 0_residue_codes_rigid.txt: Residue codes that impose full rigidity.
• 0_residue_codes-H+S_rigid-rest_flexible.txt: Residue codes for partial rigidity.
• 0_actin_orig.pdb: Original (start) structure of actin used in part II.
• 0_actin_target.pdb: Target structure for flexible fitting.

• 0_rnap1.pdb: atomic structure of RNA polymerase in "closed" conformation.
• 0_rnap2.situs: simulated EM map of RNA polymerase in "open" conformation.
• run_tutorial.bash: Bash shell script containing all commands of this tutorial (parts I and II).

In the following, we will use the first two files to flexibly fit lactoferrin as controlled by various residue constraint files that impose varying degrees of rigidity (see ddforge user guide). The user can compare all generated files to the files in the "solutions" directory. (The four files highlighted in brown color 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 work flow is indicated by brown arrows. The optional rebuillding of truncated side chains with the third party SCRWL4 tool is shown in dark blue. Visualization (orange) for the rendering of the data requires a molecular graphics viewer (we use here the VMD graphics program, Chimera and Sculptor also support Situs format). 

Standard EM formats are supported and are converted to cubic lattices in Situs format. This is done with the map2map utility. Subsequently, the data can be inspected and, if necessary, prepared for the fitting using a variety of visualization and analysis tools. The ddforge tool requires one volume and one PDB structures for the fitting, where all density must be accounted for. Atomic coordinates in PDB format can be transformed to low-resolution maps, if necessary, and vice versa, to allow docking of maps to maps or structures to structures. The resulting docked complex can be inspected in the graphics program.

The ddforge tool requires that all density must be accounted for by the fitted PDB file. Situs provides a number of map tools to adjust the map volume. For an example how this is done, we refer to the classic Situs tutorial and Fig.1 of Wriggers et al. (2011).

Before we start fitting the original lactoferrin structure to the target map, it is important 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. For an illustrative example, see the actin fitting example in part II of this tutorial.

This example runs ddforge to fit the PDB structure "0_lactoferrin_orig.pdb" into the EM map "0_lactoferrin_target.situs", and uses the residue data from the file "0_residue_codes_flexible.txt".  The remaining parameters used here are:

    1:    minimum displacement between output conformations (Angstrom)
    7:    map resolution (Angstrom)
    12:  density threshold
    50:  damp/drag ratio
    0:    side-chain rebuild flag (0 -> none)
    10:  force-field cutoff distance (Angstrom)
    1:    maximum displacement per step (Angstrom)

Secondary structure can be assigned with tools like DSSP. Setting up residue data files for large PDBs might become labor intensive. You can use the above templates as a start for other applications. Alternatively, we recommend to write a script to generate a template for further editing. Here we show such a script written in Mathematica, but a user can probably translate this to a preferred scripting language without much difficulty:

The first line specifies the chains and number of residues in the PDB. The one above is for a particular example of a PDB with a single chain called A which has 691 residues. If you had, say, 2 chains, A and B, and chain B has 200 residues, you would write instead: AllChains = {{"A", 1, 691}, {"B", 10, 200}};

If you have low-resolution (worse than 10A) maps without interior detail, you may be interested in the original feature-based flexible fitting approach we initially developed with Joachim Frank and Seth Darst in the early 2000s. Part II of the tutorial shows how to assign simulated features to the data which can then be used for flexible registration. Moreover, to improve the stereochemical quality, it is possible to constrain the distances between the features to reduce the effect of noise and other experimental limitations. 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 low-resolution applications are demonstrated in the Flexible Docking Tutorial, Part II.