Distance restraints

Prepare for output of X-PLOR NOE restraints file.

Requirements

• The dictionary containing X-PLOR-type nucleus designations has been used. If not, then some of these procedures will not work.

• As many crosspeaks as possible have been assigned in the NOESY spectra. Most small crosspeaks due to noise and other artifacts have been deleted.

• Crosspeaks must have been extracted everywhere, and there must be no spuriously split crosspeaks. For the crosspeak integration to work properly, it is vital that when two or more crosspeak overlap, they should all have been extracted, so that ANSIG knows about them and their estimated positions. The integration procedure will then take the overlap into account, even if one or more of the crosspeaks have not been sequence-specifically assigned. This holds also for crosspeaks that are close to artifacts, such as t1 noise. In such cases, one should place crosspeaks close to the proper crosspeak in order to mask out the artificial intensity due to the noise.

  The above requirement does not hold for regions that have been avoided completely, for instance if no crosspeaks at all have been extracted in the aliphatic-aliphatic region.

• Stereospecific assignments (if any) have been made and the assignments for the crosspeaks have been modified accordingly. Currently, there is no procedure to perform stereospecific assignment within ANSIG. The needed information must be gotten manually from ANSIG (NOE intensities and J-coupling constant measurements) and the stereospecific assignments made in some other way (using another program, or by qualitative arguments).

• The description given here assumes that a NOESY spectrum or spectra recorded at one single mixing-time will be used for deriving distance restraints. No AL procedure to deal with a build-up series of NOESY spectra has been written yet.

Warning!

Create copy of the crosspeaks file to work on.

A copy of the current crosspeak file is created, and switched to. The original crosspeak file is left as it is. All further modifications of the crosspeak file which this procedure executes are made to the copy, not the original.

Switch on/off NOESY spectra to use.

Before making the X-PLOR NOE restraints file, be sure that only the relevent NOESY spectra are switched on for the compilation of the NOE list in ANSIG. Check which spectra are currently switched on with the command SHOW NOE. If you need to change anything, use the command

ANSIG> SET NOE each_noesy_spectrum < ON | OFF >

where the last parameter in the command is either ON or OFF. Note that this switch is not persistent; next time you start up ANSIG you will have to do this again. It is a good idea to give this command in the initialization file.

Integration.

It is necessary to re-integrate all crosspeaks in the NOESY spectra before making an X-PLOR NOE restraints file. The intensity values produced by the crosspeak extraction (if used) are unreliable and merge, delete and move operations done on the crosspeaks usually invalidate the intensity values.

To re-integrate all crosspeaks in the NOESY specta, first compile the AL procedure integrate_spectrum:

ANSIG> COMPILE {lib}integrate_spectrum.al

Then execute the procedure for all NOESY spectra:

ANSIG> integrate_spectrum (each_noesy_spectrum)

This operation takes a while to finish, since the matrix file has to be accessed for each crosspeak.

Distance corrections and trivial distance restraints.

A decision must be made whether to use distance corrections for NOEs involving non-stereospecifically assigned protons. This depends on what method of averaging is used in the X-PLOR protocols. If 'center' averaging is used then distance corrections must be made. This is equivalent to the use of pseudo-atoms. The distance corrections are given in the dictionary, and are switched ON by default. If 'R-6' averaging is used instead, then distance corrections are not needed, and the addition of a distance correction should be switched off using the command.

ANSIG> SET CORRECTIONS OFF

If needed, then it is a good idea to put this into the initialization file.

A distance restraints derived from NOEs may be clearly trivial in the sense that it specifies an upper distance which is longer than is possible in any conformation of the intervening covalent structure. This typically may occur between intra-residue vicinal protons, e g HN/HA protons.

It is possible to switch off the output of trivial distance restraints to the X-PLOR file. This requires that the dictionary file contains specifications of trivial distances. It is possible to specify only intra-residue trivial distances.

To switch off the output of trivial distance restraints, use the command:

ANSIG> SET TRIVIAL OFF

Note that the default is ON.

Reduce methyl intensities.

When using the intensity-to-distance mapping method, then intensities for the methyl protons should first be reduced by a factor of 3. This is done by the AL procedure reduce_methyl, which relies on information given in the dictionary about which nuclei are methyls. The standard dictionary contains this information, but this should be checked if there have been modifications or additions made to it.

First compile the AL procedure reduce_methyl:

ANSIG> COMPILE {lib}reduce_methyl.al

Then execute the procedure for all NOESY spectra:

ANSIG> reduce_methyl (each_noesy_spectrum)

Note: An alternative way of dealing with the problem of higher apparent methyl intensities is to add a further 0.5 Å to the distance restraint. This can be done by using a dictionary with such distance corrections, for instance amino_acids_15N_13C_corrmethyl.dic. If this method is used, then the AL procedure reduce_methyl should not be used.

Calibrate the intensity classes

There is currently no AL procedure to calibrate the intensity classes automatically. This must be done by manually looking at selected crosspeaks within unambiguous secondary structure and deducing the limits for the relative intensities for each class. This step should be done before step 4 because some of the crosspeaks whose assignments are removed in step 4 may be of use in the calibration.

Modify non-stereospecific assignments

Assignments in ANSIG are usually made such that for non-degenerate methylenes (and similar prochiral groups) the two different nuclei are given the designations HB1 (upfield) and HB2 (downfield). This fits in with how the assignment table in ANSIG works. However, if an NOE restraints list is made based on these assignments, then it is implicitly assumed that stereo-specific assignments have been made.

This is not the case if a so-called floating-assignment protocol is used, an approach which ostensibly determines stereospecific assignments automatically as part of the structure determination. The most commonly used protocols do not use the floating-assignment method.

The AL procedure modify_nonstereo will convert those NOEs which involve non- stereospecifically assigned protons to involve instead the corresponding nucleus group, as defined in the dictionary. The procedure first reads an input file in which all stereospecific assignments have been listed. Those NOEs are then avoided by the procedure. All else are processed.

Important note: The procedure modify_nonstereo will go through all spectra used to compile the NOE list, and will remove all connections (Fdim connections, symmetry, correspond) from crosspeaks involved in NOEs that are modified. This procedure must therefore only be executed while using a temporary copy of the crosspeak file.

The second argument to the procedure (coalesce) is a flag which determines what happens to pairs NOEs of nearly equal intensity that involve both of two non-stereospecifically assigned protons. Either both are kept, or the two are represented by one single NOE.

The reason for keeping both is that since both give the same distance restraint ranges, it is not necessary to know which is which. Since both NOEs are observed, both distance restraints should be satisfied. Using only one NOE to represent both may mean that a structure is obtained which satisfies only one restraint, not both.

On the other hand, if the NOESY spectra have even a small amount of spin diffusion, then the intensities involving methylenes and pairs of methyls (the groups most often relevant to this problem) are those that will be evened out, although the actual distances should give NOEs of different intensity. In this is the case, then using one distance restraint to represent both NOEs is the proper thing to do.

If the NOEs involving the two non-stereospecifically assigned protons are of different intensity, giving different distance restraints, then only the strongest NOE is kept, regardless of the 'coalesce' flag value.

Note that the side-chain amide protons of Gln (HE21 and HE22) and Asn (HD21 and HD22) usually are stereospecifically assigned, and so should also be included in the input file for modify_nonstereo.

First prepare the file giving the stereospecific assignments:

! stereospecific assignments, examples

17 HB1 HB2
23 HD1# HD2#  ! Leu
45 HD21 HD22  ! Asn
67 HD1 HD2    ! Tyr, with resolved and stereo-
67 HE1 HE2    ! specifically assigned HD and HE

Then compile the AL procedure modify_nonstereo.al:

ANSIG> COMPILE {lib}modify_nonstereo.al

Then execute the procedure giving the stereospecific assignments file as input argument.

ANSIG> modify_nonstereo ("stereospecifics.dat", true)

The second argument is a logical value which determines how pairs of NOEs involving both of two non-stereospecifically assigned protons are dealt with. If true, then the NOE restraints will always be coalesced into one single restraint. If false, then in the case of equal strength of both restraints, then both are kept.

For example, if the two methyls of a Val have NOEs to a given proton, then both these NOE distance restraints may be kept if both are of equal strength, and no distance corrections need be added. If the NOEs are of unequal strength, then only the strongest is kept, and a distance correction has to be added since it is not know which of the two methyls are responsible for this NOE.

Finally, to produce the X-PLOR NOE distance restraints file, give the command:

ANSIG> NOE XPLOR filename

Note: X-PLOR is spelled XPLOR in this command.

The file produced can be used directly in X-PLOR.