Loopy

Building loops using structural and density information

• First it tries to find likely candidates for only the CAs of the residues. To find these likely CAs, it takes a quatrapeptide and generates a large number of possible positions in a shell of CA-CA distance to produce a pentapeptide. Next it uses a likelihood table for the angles in the resulting pentapeptides, and the density at the generated positions to determine the set of best CAs. By iterating over the number of missing residues, this procedure builds a tree of possible CA-CA paths which would connect the fragments.
• The tree of possible paths is then pruned to remove unlikely paths and keep the most likely ones. This is done in the following steps:
  1. Since no restriction was placed on the end position of the loop, the first pruning is done on the distance between the loop and the connecting fragment. Loops are kept, if the distance between the end CA of the loop and the connecting CA of the fragment is approximately equal to the CA-CA distance.
  2. (obsolete) Depending on the direction in which the loop was build, the N or the C of the connecting fragment is known. We use this information to check the CA_fragment-CA_loop_N angle or CA_fragment-CA_loop_C respectively.
  3. Though the structural likelihood is used in the direction of loop building, no information was used on the structural likelihood of the loop and the connecting fragment. In this step the most likely loops according to the structure are kept.
  4. If you want, you can prune the tree even further by keeping only those loops with a high average density at the suggested CA positions
  5. Next the peptide planes are determined. We use the fact, that the atoms between CA and CA lie in a plane, and the relative position of N, C, and O atom are known. By rotating around the CA-CA bond, the plane with the best density correlation for the main chain atoms (and worst density correlation outside the plane) is determined. For non-GLY residues, the density correlation at the CB is used as well.
  6. Finally, loops that not comply with the ramachandran plot are removed. (We used the table as given by K. Kelly where we removed those (φ,ψ) combinations with a value 0)
• When all the loops are build (so if chosen, in both direction), the loops are ordered (in descending order) according to the density correlation at the main chain atoms (including CB if present) or the correlation of the side chains, or a combination of both. If the number of loops exceeds the chosen number only the best are saved to file.

Protocol

Job title

Title for the current experiment

Experimental data

Select whether to use a map or an mtz file. In the case of an mtz file, the program will use fft to compute the corresponding map.

Files

Input map

Input map to use

MTZ

Mtz file to use. F and PHI are used to compute the corresponding map using fft. We need to save this file, since we need to reread the map more than once.

Input pdb

Input pdb for your protein. Please, remove residues which you would like to rebuild from this file. This frontend of loopy will not rebuild any residues.

Name for first loop pdb

The name of this file is used as a format to determine the names of the other loops to save

Number of loops

Select the number of loops you'd like the program to save. It might very well be that the number of loops left after pruning is less, than this number. In that case the number of loops saved, will be less than you asked for. If no loops are found at all, twiddle with the parameters, specifically those in the folder "Selecting best CAs"

Crystal Parameters

Definition of loop

N-term anchor

Anchor residue of a fragment on the N terminus side of the protein. Note that if you want to rebuild some residues, you need to remove them from the pdb file

C-term anchor

Anchor residue of a fragment on the C terminus side of the protein. Note that if you want to rebuild some residues, you need to remove them from the pdb file

Loop length

Number of residues in the loop including the two anchor points

Loop sequence

Sequence of amino acids (one letter code) of the residues in the loop including the two anchor points.

Build both ways

If selected (default) trees of possible loops are generated starting from the N terminus anchor, and from the C terminus anchor. The best loops to save are selected from the combined set. Since the quatrapeptides from either end of the fragments will in general differ, just as the map, starting from a different anchor will influence the loops generated.

Build towards C-terminus

If you didn't select to build both ways, you can indicate whether you want to build the tree towards the C terminus of the protein, or towards the N terminus

Selecting best loops

Deviation distance loop connection

The distance between the end CA of the loop and the connecting CA of the structure should be approximately equal to CA-CA distance. Set the allowed error in the distance.

Threshold density correlation CAs

After pruning on the distance, the next step is to select the best trees based on the density correlation of the CAs. This number sets the number of best loops kept based on the density correlation of the CAs only.

Structural threshold

You can prune on the structure of the end CA of the loop and the connecting quatrapeptide. Set the threshold for the minimal value for the log likelihood of this structure

Minimum for this stage

Set this value, if you want to ensure to keep at least a certain number of loops after pruning on the structure... overruling the structural threshold if necessary

Maximum for this stage

Set this value, if you want to ensure that the number of loops doesn't exceed a certain amount after structural pruning... keeping only those with the highest structural likelihood

Main chain density correlation

After pruning on the structure, the peptide planes for all residues in the selected loops are determined. The loops are sorted to the best density correlation of the main chain atoms (including Cb for non-GLY). This threshold sets the number of best loops kept

Weight main chain

Finally the best loops are selected by determining the density correlation of the main chain atoms (including Cb if present) and the correlation of the side chains. You can use this weight to give the main chain correlation more or less impact.

Weight side chain

Finally the best loops are selected by determining the density correlation of the main chain atoms (including Cb if present) and the correlation of the side chains. You can use this weight to give the side chain correlation more or less impact.

Selecting best CAs

Likelihood threshold

This is the threshold for the log likelihood of a CA to represent the fifth CA of a peptapeptide, based on density correlation, CA-CA distance, and structure.

Weight distance

Weight for the distance likelihood

Weight density

Weight for the likelihood of the density correlation

Weight structure

Weight for the structural likelihood

Structure table to C

Filename for the probability table for the angles and dihedral angles of a pentapeptide in the direction of the C terminus

Structure table to N

Filename for the probability table for the angles and dihedral angles of a pentapeptide in the direction of the N terminus

Minimum distance CA

Measure for the minimal distance between CAs from the same shell. The CA with the best likelihood is kept.

Maximum number of CAs

Maximum number of CAs from each shell to keep. Note: The CAs kept will all be used as a new suggestion for the current residue in the loop, and thus as a new node in the tree. The number of possible loops generated will expand exponentially with this number.

Force minimum number of CAs

Force a minimum number of CAs in a shell to be kept, even if the likelihood is less than the threshold set. This makes the loop building a bit more flexible in low density areas, or for pentapeptide structures which occur less often.

Generating CAs

Select generation CA shell

Default a shell with a uniform and regular distribution of CAs at exactly CA-CA distance is generated. You can also choose for a uniform and random distribution of the CAs. In that case the shell is generated with a given thickness

Number of CAs

Number of CAs generated within a shell. In the case of a regular distribution this number is rounded downwards to the closest Fibonacci number.

CA-CA distance

Distance to use between successive CAs.

Shell thickness

(random shell only) Thickness of the generated shell of CAs.

SD CA-CA distance

(random shell only) We assume that the probability for the CA-CA distance is described by a Gaussian. With this value you can set the standard deviation fo the Gaussian function.

Keep CAs with negative density halfway

Due to the structure of a peptide, we expect the density correlation halfway between successive CAs to be positive. A quick first selection of CAs from the shell is thus (apart from the density correlation at the generated CA) based on the density correlation midpoint. Default for this option is false.

Density Handling

Interpolation method

Choose the interpolation method used to determine the density correlation from the map. The quick, but less accurate option is Cubic Interpolation. More accurate, but significantly slower is Best, which is based on a gaussian density correlation function to similate the shape of an atom. Note: the density correlation is determined many times, thus the difference in speed between the options can be huge. The side chain correlation at the end is always determined using the gaussian version.

Atom radius

Radius used to determine the density correlation

B factor

At the moment the values for the b-factor in the pdb are ignored. The value set, will be used for all atoms

Remove atoms by factor

To avoid overlaps between the generated loop and the protein structure in the pdb, atoms in the pdb (apart from dummies, or residues in chains consisting only of main chain atoms) are removed from the map. This is done by flipping the density in the map to negative values at the position of these atoms. With this factor you can set the factor with which the density is changed

Density threshold residues

Threshold for the density correlation of residues after loop building. This is used to check overlap between the loop and possible fragments of main chain atoms in the pdb.

Density threshold dummies

Threshold for the density correlation of dummies after loop building. This is used to check overlap between the loop and possible dummy atoms in the pdb.

Log files of Loopy

Message level

Level of the messages to be written to file. (Value from 0 till 9)

Abort level

If a message of this level is encountered, terminate the program. Standard values are 7 or 8

Message file

Name for the message file (plain text) of Loopy.

XML output file

Name for the XML message file (xml format) of Loopy.