CYANA Macro: noeassign

Parameters

peaks=string (required)

format=string

(default: none)

prot=string (required)

cycles=integer range

(default: 1..7)

combination=integer range

(default: 1..2)

keep=string

(default: none)

confine=real

(default: 1.0E10)

calculation=string

(default: structcalc)

autoaco

shiftassign

multiple

stereoexpand

details

Description

Performs a complete structure calculation with several cycles of automated NOE assignment (Herrmann et al., 2002) according to the following parameters:

peaks

Names of the input NOESY peak lists. Multiple peak list names must be separated by commas without blanks before or after a comma. Peak list names with the extension .xpk refer to NMRView peak lists, otherwise XEASY format peak lists with default extension .peaks are assumed.

format

Order of the dimensions in each of the peak lists given by the parameter peaks. Multiple format declarations, given in the same order as the corresponding peak list names in the peaks parameter, must be separated by commas without blanks before or after a comma. The order of dimensions of a peak list is given by a string with one of character for each dimension. H and h denote ¹H dimensions, N or C stand for the dimension of the ¹³C or ¹⁵N nucleus that is covalently bound to the proton of the H dimension in 3D and 4D NOESY spectra, and n or c stand for the dimension of the ¹³C or ¹⁵N nucleus that is covalently bound to the proton of the h dimension in 4D NOESY spectra. For instance, the format string ChH for a 3D NOESY spectrum indicates that the three dimensions in the peak list correspond, in this order, to ¹³C, the “free” ¹H, and the ¹H that is bound to the ¹³C of the first dimension. The format parameter is optional if the order of the dimensions in the peak lists can be determined otherwise, i.e. if either all peak lists are 2D, or if format declarations are included in the peak lists with “#CYANAFORMAT” entries, or if the program can unambiguously deduce the correct ordering of the dimensions from assigned peaks in the peak lists. In general, it is most convenient to declare the order of the dimensions in the peak list by a “#CYANAFORMAT” statement.

prot

Names of the input chemical shift lists. Multiple chemical shift list names must be separated by commas without blanks before or after a comma. The default extension for chemical shift list names is .prot. Each chemical shift list is used for the corresponding peak list given in the peaks parameter. If less chemical shift lists are given than there are peak lists, then the last chemical shift list is used for all remaining peak lists. In particular, it is possible, and recommended, to use a single chemical shift list for all peak lists.

cycles

Cycles of automated NOESY assignment and structure calculation that are performed, given as an integer range. By default, seven cycles numbered 1–7 are performed, followed by a final structure calculation using the NOE assignments of the last cycle. Cycles are skipped if their output structure (cyclen.pdb), or the output structure of a later cycle, already exists. This allows to automatically continue an interrupted noeassign calculation with the next unfinished cycle. To repeat a complete calculation, it is necessary to remove the output files of the previous run, for instance with the cyanaclean command.

combination

Cycles of automated NOESY assignment in which constraint combination is applied, given as an integer range. Since the largest numbers of incorrect distance restraints occur in the first cycles, and because constraint combination entails a (temporary) loss of structural data, constraint combination is typically applied in the first two cycles only.

keep

Name of a CYANA macro or command that selects those assigned peaks whose assignment should be kept unchanged during automated NOE assignment. By default, i.e. if this parameter is absent, the program will discard any preexisting NOE assignments in the input peak lists, and will try to assign all peaks. For example, defining a command

	  subroutine KEEP
	    peaks select "*, *"
	  end

and calling the noeassign command as

	  noeassign … keep=KEEP …

will keep all peak assignments in the input peak lists fixed and lets the program search for new NOE assignments only for previously unassigned peaks.

confine

Maximal effective distance restraint violation contributing to the target function with violation confinement in cycles 1–2. See variable viocap for details. The confine value is assigned to the variable viocap in cycles 1–2. In subsequent cycles violation confinement is not applied.

calculation

CYANA command used to execute the structure calculation in each cycle. The standard macro structcalc is used is used by default. A user-defined alternative command must understand the same parameters as structcalc.

autoaco

Option to specify the use of temporary torsion angle restraints that favor the allowed regions of the Ramachandran plot (Laskowski et al., 1996) and the staggered rotamer positions for torsion angles between atoms with four covalent bonds, e.g. tetrahedral carbons. The temporary torsion angle restraints are generated with the commands ramaaco and rotameraco in the intermediate cycles, and with the commands ramaaco minimal and rotameraco for the final structure calculation. Temporary torsion angle restraints are applied in the initial and intermediate stages but not in the final stage of the standard simulated annealing schedule with the command anneal.

shiftassign

Option to enable the assignment of missing chemical shift during automated NOE assignment. In this case a new chemical shift list, cyclen.prot, is produced and used in each cycle. The reliability of new chemical shift assignments made in this way should be evaluated critically by the user.

multiple

Option to allow for ambiguous distance restraints also in the final structure calculation. By default, only unambiguous distance restraints are used for the final structure calculation. Ambiguous distance restraints from the last intermediate cycle are split into multiple unambiguous distance restraints, or discarded.

stereoexpand

Option to use the command distance stereoexpand instead of the standard command distance modify to account for the absence of stereospecific assignments.

details

Option to produce, in addition to the normal output, also assigned peak lists from each, not only the last, cycle. In addition XEASY assignment (.assign) files are produced if XEASY format peak lists are used.

Algorithm overview

The algorithm for automated NOE assignment is a re-implementation of the former CANDID procedure (Herrmann et al., 2002) on the basis of a probabilistic treatment of the NOE assignment process. The key features of the algorithm are network anchoring to reduce the initial ambiguity of NOESY peak assignments, ambiguous distance restraints to generate conformational restraints from NOESY cross peaks with multiple possible assignments, and constraint combination to minimize the impact of erroneous distance restraints on the structure. Automated NOE assignment and the structure calculation are combined in an iterative process that comprises, typically, seven cycles of automated NOE assignment and structure calculation, followed by a final structure calculation using only unambiguously assigned distance restraints. Between subsequent cycles, information is transferred exclu¬sively through the intermediary 3D structures. The molecular structure obtained in a given cycle is used to guide the NOE assignments in the following cycle. Otherwise, the same input data are used for all cycles, that is, the amino acid sequence of the protein, one or several chemical shift lists from the sequence-specific resonance assignment, and one or several lists containing the positions and volumes of cross peaks in 2D, 3D or 4D NOESY spectra. The input may further include previously assigned NOE upper distance bounds or other previously assigned conformational restraints for the structure calculation. In each cycle, first all assignment possibilities of a peak are generated on the basis of the chemical shift values that match the peak position within given tolerance values, and the quality of the fit is expressed by a Gaussian probability, P_shifts. Second, the probability P_structure for agreement with the preliminary structure from the preceding cycle, represented by a bundle of conformers, is computed as the fraction of the conformers in which the corresponding distance is shorter than the upper distance bound plus the acceptable distance restraint violation cutoff. 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 short-range distances. The network anchoring probability P_network that the distance corresponding to an assignment possibility 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-dimensional structure. Contributions to the network anchoring probability for a given “current” assignment possibility result from other peaks with the same assignment, from pairs of peaks that connect indirectly the two atoms of the current assignment possibility via a third atom, and from peaks that connect an atom in the vicinity of the first atom of the current assignment with an atom in the vicinity of the second atom