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== Parameters ==

; alignfactor=''real'': (default: 0.5)
; matchfactor=''real'': (default: 0.5)
; violation=''real'': (default: -1.0)
; probability=''real'': (default: 0.2)
; quality=''real'': (default: 0.5)
; elasticity=''real range'': (default: 1.0..1.0)
; confidence=''real'': (default: 1.0)
; supportweight=''real'': (default: 1.0)
; prefer=''integer'': (default: 999999)
; interrange=''integer range'': (default: 0..)
; unassigned=''real'': (default: 0.1)
; short
; changevol

== Description ==

The '''assign''' command performs automated assignment of the NOESY
cross peaks on the basis of the given chemical shifts, knowledge of
covalently constrained short distances, and the selected 3D conformers,
if available. The '''assign''' command is used in the '''noeassign''' macro
to implement a combined automated NOESY assignment and structure
calculation strategy.

Input data:

Required input data consists of unassigned (or assigned) NOESY
peaks from one or several peak lists, and one or several chemical
shift lists. Optional input data comprises a group of selected
conformers and a list of covalently constrained short distances. To
each input peak an upper distance bound must have been attributed,
for instance using the '''peaks simplecal''' command or the '''calibration'''
macro that convert peak intensitites or volumes into distance bounds.

Output data:

Output data comprises assignments made by the '''assign''' command for
the peaks that were NOT selected in the input peak lists, as well as a
report including details on the assignment of each individual peak and
a summary table. Peaks that were selected on input are not modified. If
peaks are assigned and unselected on input, the report also provides
a comparison between the input assignment and the new assignment made
by the '''assign''' command that overwrites the input assignment.

Assignment strategy:

First all assignment possibilities of a peak are generated on the
basis of the chemical shift values that match the peak position
within the tolerance defined by the '''tolerance''' variable. Second,
the probability for agreement with the bundle of selected conformers,
if present, is computed as the fraction of the conformers in which the
corresponding distance is shorter than the upper distance bound plus
the acceptable ''violation'', and assignment possibilities for which the
product of these two probabilities is below the required ''probability''
threshold are discarded. Third, each remaining assignment possibility
is evaluated for its network anchoring, i.e., its embedding in the
network formed by the assignment possibilities of all the other peaks
and the covalently constrained distances. The network anchoring
probability that the distance corresponding to an assignment is
shorter than the upper distance bound plus the acceptable ''violation''
is computed given the assignments of the other peaks but independent
from knowledge of the three-dimensionl structure. Only assignment
possibilities for which the product of the three probabilities is
above the required ''probability'' threshold, are accepted. Next the
overall quality Q of the assignment of a peak is computed from the
probabilities of its individual accepted assignment possibilities. The
overall quality of a peak assignment is always at least as large as
the highest probability of an accepted assignment possibility. Peaks
are kept assigned only if their quality exceeds the ''quality'' cutoff.

Example assignment report for a peak:

Peak 165 from c13.peaks (8.72, 4.11, 59.86 ppm; 3.08 A):
2 out of 4 assignments used, quality = 0.97:
* H ILE 64 + HA ILE 63 OK 90 99 100 91 2.1-2.3 1260=69, 63/50=24...(10)
H ILE 63 + HA ILE 63 OK 71 71 100 100 2.8-2.8 3.0=100
H SER 43 - HA ILE 63 far 0 95 0 - 6.4-9.0
H ALA 22 - HA ILE 63 far 0 99 0 - 9.9-14.6
Violated in 0 structures by 0.00 A.

* Line 1: Peak number, peak list, peak position, upper distance bound.
* Line 2: Number of used assignments, number of assignment possibilities,
overall quality of the peak assignment (0..1). Quality values below
the ''quality'' cutoff are marked as "low quality", and the peak remains
unassigned.
* Lines 3-7: Individual assignment possibilities
* Flag that indicates the input assignment, if present, by a '''*''' if
it is among the used assignments, or by a '''!''' otherwise.
* First atom, identified by its name, residue name, and residue number
* Flag: '''+''', used assignment; '''-''', assignment possibility not used
* Second atom, identified by its name, residue name and number
* Decision on assignment possibility:
'''OK''', good assignment with probability above the ''probability'' cutoff
'''far''', structure based probability too low
'''lone''', network anchoring based probability too low
'''poor''', individual probabilities ok but overall probability too low
* Overall probability for the assignment possibility (%)
* Probability for match between peak position and chemical shifts (%)
* Probability for agreement with input structure bundle (%)
* Probability derived from network anchoring (%)
* Minimal and maximal distance in the selected conformers (Angstrom)
* Most important individual contributions to the network anchoring
based probability, ordered by decreasing size. The number after the
equal sign is the probability in percent for the contribution
identified in front of the equal sign, as follows (only the first
three possibilities appear in the example above):
''real'': covalently constrained distance shorter than ''real'' A.
''integer'': peak number of a (symmetrically related) peak with the
same assignment
''integer''/''integer'': numbers of two peaks that relate the two atoms
of the present assignment through a third atom
''integer''/''real'': peak with number ''integer'' connects the first atom
to a third atom whose distance from the second atom is
covalently restrained to be shorter than ''real'' A.
''real''/''integer'': peak with number ''integer'' connects the second
atom to a third atom whose distance from the first atom
is covalently restrained to be shorter than ''real'' A.
~''integer'': The peak with number ''integer'' connects two atoms that
covalently restrained to be less than x A from the first
and second atom of the present assignment possibility,
respectively.
For reasons of space, only the first few contributions are printed.
An ellipsis "..." followed by the total number of contributions
in parenthesis indicates that not all contributions with probability
greater than 1% are printed.
* Line 8 (last line): Number of conformers in which the upper distance
limit of the ambiguous distance restraint formed by the accepted
assignments (marked by '''+''' in lines 3-7) is violated by more than
the ''violation'' threshold, and the average size of the violation.

Covalently contrained distances:

The covalently constrained short distances are normally taken from
distance restraints with weight zero, which can be obtained, for
instance, by analyzing a bundle of randomized conformers with the
'''distance short''' command, as implemented in the '''noeassign''' macro. If
no distance restraints with weight zero exist, the short distances
are calculated internally from the select conformers (which should
be randomized), if available and if ''violation'' is negative, or by
an analytical calculation otherwise.

Elasticity of upper distance bounds:

When searching for peak assignments the algorithm can adapt individual
upper distance bounds in the input peak lists by a factor within
the allowed ''elasticity'' range. An individual upper bound can be
increased if a slight violation of the original upper distance bound
can be avoided by the increased distance limit in at least 80% of
the conformers. An individual upper bound can be decreased if the
actual distances in the input conformers are consistently shorter
than the upper distance bound. By default, there is no "elasticity"
of the upper distance bounds, i.e. the input distance limits are used
without change. If an upper distance is changed, its modified value
is indicated in the first line of the report on the assignment of the
peak. The additional option '''changevol''' can be used to correct peak
volumes according to the internal change of the corresponding upper
distance bound using an inverse sixth power relationship.

Additional control parameters:

The probability for the chemical shift matching is calculated
using the tolerance values multiplied by ''matchfactor''. A smaller
''matchfactor'' implies a higher weight for good agreement between the
peak coordinates and the chemical shifts. The mutual alignment of peaks
is controlled by the variable '''tolerance''', and the probability for
network anchoring is calculated using the tolerance values multiplied
by ''alignfactor''. A smaller ''alignfactor'' implies a higher weight for
good mutual alignment between peaks with assignment possibilities to
the same atom(s). When calculating the network anchoring probability
of a given peak assignment, the probabilities of other aligned peaks
may be scaled by a ''confidence'' factor between 0 and 1. Chemical
shift assignments with an attached chemical shift error larger than
the ''unassigned'' cutoff are treated as "unassigned" when determining
the initial assignment possibilities of peaks: Only one of the two
atoms of an assignment may be "unassigned", and, if in addition the
'''short''' option is set, only short-range assignments for covalently
constrained distances are considered.

Symmetric homodimers:

The '''assign''' command provides special features for symmetric homodimers
that can be defined with the '''molecules define''' command. In the case
of a homodimer, only assignments with the first atom in the first
monomer are made. The corresponding symmetric distance restraint can be
added afterwards with the '''molecules symmetrize''' command. Homodimer
assignments are restricted to be only intramolecular or only
intermolecular for peaks with (XEASY) color codes 8 or 9, respectively.
Furthermore, intermolecular homodimer assignments between residues
i and j are considered only if |i-j| is within the ''interrange''.
Intermolecular assignments of a peak are also excluded if the peak
has at least one intramolecular assignment between residues i and j
with |i-j| smaller than ''prefer''.

Further reading:

* Herrmann et al. J. Mol. Biol. 319, 209-227 (2002).
(Note that the algorithm implemented in the '''assign''' command differs
significantly from the original CANDID algorithm described in this
publication.)
* Guntert. Meth. Mol. Biol. 278, 353-378 (2004).
* Guntert. Prog. NMR Spectrosc. 43, 105-125 (2003).
* Jee & Guntert. J. Struct. Funct. Genom. 4, 179-189 (2003).

== See also ==

* [[noeassign]]
* [[tolerance]]

CYANA Commands: assign - Revision history

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