Special tools are provided to handle peptides and
proteins. This can be done on two levels of complexity. On
the coarser level entire amino acids are treated as units.
These are represented as single points, but also carry
information about torsional angles. Such entries must be
handled with a separate set of tools which are provided in
the Calf building module (option c). On the finer level, all
atoms are included and the normal modelling tools apply. In
addition special testing and refinement utilities are
provided (option p). These two types of structures can be
mutually generated from each other (options k, q, a and b).
When building Calf structures one uses a similar
repertoire of activities as in small-molecule building, at
least for the basic steps. However, a couple of
possibilities have no small-molecule counterpart. In
addition to the pure building facilities, a couple of
auxiliary options are placed within this menu.
For a set of consecutive residues a database can be
searched for conformations fitting the given positions
optimally. Optionally the agreement of the backbone dihedral
angles can also be included in the score function.
The database is built up by reading in protein structures
from disc files. Only coordinates of the Calfs and the values
for the backbone dihedrals are kept.
From a known protein structure a structural model for a
homologous protein can be built, provided a sequence
alignment is given. The alignment must be given in a file in
which each line is interpreted as single letter code
sequence of the original protein or of the homologous one,
provided the line starts with either #1 or #2 respectively.
All other lines are interpreted as comments. In case of
deletions or insertions, missing residues must be indicated
by a dot. For deletions the program simply connects the two
ends of the last and first residues present. Insertions are
positioned near a circle of appropriate size. It is
understood that these regions are subjected to subsequent
energy minimization within the Calf force field to generate
acceptable geometries. The program automatically generates a
user set which contains the residues in the homologous
structure, which may be kept fixed under a subsequent
minimization. This set must be activated (definition of
fixed residues) prior to minimization.
A selection of options useful in positioning a Calf
structure into a electron density map.
Real space refinement to fit segments of all-atom
(protein) structures to a given electron density map can be
performed here.
This tool offers a set of options which are specific to
biopolymer molecules (mostly proteins). Structures can be
tested, refined, forged and displayed in color coded
fashion.
Proteins in all atom form can be modified here with respect
to side chains. Incomplete side chains can be completed.
Side chains can be replaced by others.
New water molecules can be inserted by picking anything
with the left mouse button. The new atoms appear in the
center of the visibility volume. If an other atom is nearby,
no water is inserted and a corresponding message is issued.
Picking with the middle button removes the picked atom, if it
is an isolated oxygen. Insertion is silenced if the move
option a is activated!
There are two similar menus for scanning through isolated
atoms or residues. For atoms:
There is always a currently selected atom, which carries
its label. All other isolated atoms are only marked. Picking
an isolated atom makes it the selected one. Pressing the
shift key and operating the mouse moves the currently
selected atom about.
For residues:
There is always a currently selected residue, which
carries its label at the Calf atom. By default the average B-
factor is calculated. If a map is specified, the user can
have map-gradient and RSCC (real space correlation
coefficient) values evaluated as well.
This menu facilitates the setup of an energy optimization
calculation for large polymeric systems. Automatic sets on
proteins and nucleic acid can be held fixed (stationary) or
be subjected to positional constraints as a whole. Upon
entering this option, the entries to be refined together
must all be in the active state.
Measured or modelled protein structures can be checked
here for various stereochemical and energetic properties.
Checks are performed entry-wise. Written results can be
output to a file.
Protein and C-alpha Utilities [pca]
Furthermore, dedicated tools for structure building in
protein crystallography are implemented, such as real space
refinement, structure scanning etc.
,: peptide representation
To indicate the orientation of peptide links in Calf
structures, the representation of the structure by con
necting linked Calfs is augmented by an additional line
between the calculated positions of the nitrogen and
oxygen atoms of the peptide. Furthermore, the C-alpha
to C-beta bond is shown to indicate how the side chain
branches off. The auxiliary display is initiated by
picking the structure. It is only updated during opti
mizations and when peptide links are rotated (forge).
The user can initiate an update by picking the struc
ture again. Picking with the middle button removes the
auxiliary display.
k: generate Calf structure
In Moloc, Calf structures of proteins contain more
information than just the positions of the alpha carbon
atoms. They are suited to perform energy calculations
with the peptide-mechanics force field. The present
option generates this type of structure from a full
protein entry by picking it. If the protein is incom
plete and of poor geometry, it may be advisable to uti
lize option q!
a: generate full peptide (all atoms from Calf)
By picking a Calf entry the corresponding full protein
is generated.
b: generate backbone structure from Calf
Generates the backbone of a protein by picking a Calf
structure. The backbone structures include carbonyl
oxygens and beta carbons, if present.
u: update full structure from Calf or from all-atom
fragment
The coordinates of a full protein structure are modi
fied for each residue which has been modified during a
modelling process in the corresponding Calf structure.
When updating from an all atom fragment entry, the
fragment is expected to be a polymer of which every
monomer has a corresponding one in the full structure.
The coordinates of the fragment atoms are then trans
ferred to the corresponding atom in the full structure.
Pick full structure (to be updated) first, then the
modified Calf structure or the all-atom fragment entry,
which provide the new coordinates. If residues have
been added to the Calf structure, an updated full
structure must be produced it two steps: First, gener
ate a new full structure from Calf (option a) which will
be the result. Second, update this result structure
with the original full structure, in order to transfer
the coordinates of the atoms which existed before the
addition. Heterogens are not transferred in this pro
cess!
g: geometry examination
f: forge
c: Calf building
This is the central tool to build and modify proteins on
a coarse level (C-alpha's only) where secondary-struc
ture and folding problems must be handled.
m: modifying (forging) Calf structure
A collection of tools arranged in a way to facilitate
efficient positioning of a Calf structure into a elec
tron density map, including side-chain arrangement.
r: real space refinement of protein structure
Structures (or parts thereof) are optimized with the
MAB force field, augmented by a term that locally maxi
mizes overlap between structure and a specified elec
tron density map. When maps are visualized with the t-
option, it must be remembered, that translated contours
may be visible in regions where the map is not explic
itly defined. At present, real space refinement only
works in regions, where the map values are explicitly
given.
p: protein refinement, tests etc.
This is a utility to handle all-atom protein- (and, to
some extent DNA-) structures
q: generate Calf structure (poor geometry)
If the peptide is incomplete (e.g. only Calfs known)
and the geometry of the atomic positions is poor, this
option will generate a Moloc-Calf assuming that a sin
gle consecutive chain is given.
C
a
Building [cab]
n: open new entry (Ala-Ala)
To enable starting to build a new sequence a Ala-Ala
dipeptide is generated at the center of the actual dis
play volume.
a: build sequence (add, delete residues)
When building a sequence one has to start from an exist
ing structure. Thus, before entering this option be
sure to have a starting chain (e.g. Ala-Ala).
b: make, break bonds (peptide links, S-S)
To make a link between two residues, pick the residues
to be bonded. Since most peptide links are generated
while building the chain, you will be asked whether you
need a peptide link or not. A peptide link is only added
if the existing link situation permits it.
c: change types of single residues
When a residue is picked a choice requester appears
with a list of available amino acids.
i: add isolated fragment (Ala-Ala)
At the center of the actual display volume a dipeptide
Ala-Ala is added to the picked entry.
l: add local entries
A copy of any local entry (picked first) can be taken
over to another entry (picked second). If the copy is
not repositioned (r not highlighted in the new menu),
its origin becomes invisible under the action.
d: delete sets of residues
f: forge structures
s: search database for conformations
Search database for suitable secondary structure con
formations.
o: optimize Calf structure
Invokes the Peptide-Mechanics force field. All active
Calf structures are affected.
h: homology building
To perform homology building, the Calf structure of a
known protein is needed, together with an alignment
file with the primary sequence of the protein to be
built. In this file, each line containing single letter
coding of the known structure should start with #1 ,
while lines of the unknown structures must start with
#2 . Gaps or insertions must be indicated by appropri
ately placed dots.
r: regularize Calf structure
This is a distance geometry type algorithm which
adjusts peptide link lengths and pseudo valence angles
to acceptable values. It is useful as a first treatment
of structures only given by Calf positions. However,
such structures, when read in from pdb-files must first
be changed to Moloc-type Calf structures in the previ
ous menu. After execution of the present option it may
be advisable to recalculate phi and psi from scratch,
if these values could not be taken from the input file.
j: calculate phi-psi from scratch
A new set of phi and psi values is calculated using the
geometry of the Calf positions alone. This tool may be
useful in cases where only Calf positions are given. It
is automatically invoked while generating a Moloc-Calf
structure if the corresponding full structure lacks the
necessary information (peptide links not given). The
option may reasonably be called after a regularization
step, during which the Calf geometry has changed to
some extent.
v: display of residue specific values
Residue-specific quantities can be displayed in a
color-coded form. The quantities must be scaled to
integers from zero to nine. They are entered from a file
similar to an alignment file (see homology building),
except that the #2 lines contain the integers corre
sponding to the quantity to be displayed. Of course no
dots for gaps are allowed. Colors are yellow (9,8), red
(7,6), pink (5,4), and blue (3,2). No color is given for
(1,0).
y: relabel structure
The picked Calf structure is sequentially rearranged.
This may be necessary after building, when residues are
inserted in the middle of an existing structure.
w: worry (randomize) structure
Random phi-psi values are generated that obey energy
criteria (see option s). From these values coordinates
are generated afresh.
s: set energy threshold for worry
Values for phi-psi of option w are determined by
repeated generation of random ones. From these the
energy of the phi-psi term (see Calf force field) rela
tive to the minimal energy (assumed for the alpha helix
conformation) for the given residue type is calculated.
A new random-pair conformation is accepted or rejected
by a Boltzmann type criterion in which the threshold
energy plays the role of kT.
Secondary structure search
b: build database
The database to be searched is generated from given
protein structures.
s: define search set
The set of residues for which conformation proposals
are wanted is defined here within the set menu. Only a
single stretch of consecutive amino acids can be han
dled!
w: set residue weights
For each amino acid a weight can be set (default is one)
which governs its importance in evaluating the score of
the match. For unknown loops these weights are put to
zero, but a few of the end atoms need to have nonzero
weights (e.g. three on each end).
p: set phi-psi emphasis
In case where it is important to maintain the backbone
dihedral angles, the score can be augmented by devia
tions of the angles.
d: do the search
The database defined under option b is searched for
conformations of lowest score as evaluated with the
specified weights. When the search is completed the
user is automatically dropped into the examination menu
e.
e: examine hits
The ten hits of lowest score can be examined. Choice of
? in this menu displays the scores and locations of the
matches. The number in brackets displays the phi-psi
score, if only positions contribute to the ranking
score, otherwise they show the positional score. The
display shows the chosen configuration. The original
conformation can be reset as long as the search set is
not redefined.
Build Database for Backbone Conformations
c: mca-file (Calf structures)
This file type is most suited for a quick build-up of
the database, because it contains almost no unused
information. If the user cares for a special set of
structures, it is most efficient to keep them in this
file format. A default file, called pdb.mca is avail
able in Moloc's dat directory. It can be accessed by
specifying the file name $DAT/*.
p: pdb-file
The standard Brookhaven format for proteins
w: cor-file
A format produced by the NMR software DIANA
l: local structures
This option allows to add structures which have already
been read into core memory before.
d: delete entries
The structures are kept chain-wise, such that single
chains can be removed from the database. This option
can also be used to display all the chains that are
already present.
Homology Building [bhm]
r: restore original alignment (compare next option)
If one is not satisfied with the shifts of deletions and
insertions this option restores the alignment as was
read from the file.
s: shift deletions and insertions
It may occur that visual inspection reveals that dele
tions or insertions are not appropriately positioned.
In this case this option allows to shift them in single
residue steps along the chain. To do so the longer of
the two loops must be picked. Picking with the left hand
(middle) mouse button shifts it along the chain in for
ward (backward) direction.
w: write alignment file
If an alignment has been modified by shifts of inser
tions or deletions the current alignment can be written
onto a file by choosing this option. No comment records
are generated. The records are arranged sequence-wise.
Moving a C
a
Structure [camv]
,: peptide representation (see
top Section)
a: move single residue (see "a" in
Forge)
l: turn peptide links (see "l" in
Forge)
h: drive chi angles (see "h" in
Forge)
f: forge Calf structures (see
Forge)
r: refine stretch
Here, the Peptide-Mechanics force field is invoked. The
residues to be affected are selected in the set menu.
All other residues will be flagged for being held
fixed.
Real Space Refinement [rsr]
m: choose map
If several maps have been read in, the relevant one has
to be selected here
s: choose set of atoms
For the atoms, which the user defines in this set, terms
will be added to the force field which favor placement
at positions of high electron density.
r: do refinement
A fragment of the structure will be copied in a new
entry for which a energy minimization will be made. All
atoms in the specified set and, in addition a environ
ment, defined by the environment mode and the corre
sponding parameters is also copied. The user is dropped
into the setup menu for the force field, where he can
make modifications, run dynamics or a minimization. If
large movements are envisaged it may be advisable to
set flags for preservation of the stereochemistry.
Playing with the weight of the rsr-term may also help to
speed up the minimization process. The H-bond term is
by default omitted (weight zero) but may be included as
well.
u: update structure
This option has only an effect if a fragment copy is
present. Such a copy is generated when executing option
r. The coordinates of the (not fixed) atoms of the copy
are read back to the original and the copy is deleted.
p: set parameters
Several parameters needed for the refinement can be
modified here (reasonable values are predefined). (1)
Thickness of topological layer of free atoms. (2) Dis
tance threshold for inclusion of an atom in the fixed
environment. (3) Radius of action of a map point on
neighboring atoms. (4) Threshold value of the electron
density. Points with a density below the threshold are
omitted.
e: set environment modus
Three environment modi are provided: (1) The specified
set of atoms is unchanged and completely free in the
force field treatment. (2) Around the defined set of
atoms a topological layer of free atoms (not affected
by the map energy term) is added (its depth is defined
in option p [1]) and after that an additional topologi
cal layer of fixed atoms is added. This layer has in
principle depth one, but is augmented such that mono
mers are fully included. (3) The fixed layer is not
defined by topological but by geometrical distance such
that the refined set sees its environment during the
force field calculation.
f: forge structure
The full structure only can be forged here. Forging the
fragment copy is possible in the force-field setup
menu.
g: show gradient of map
The gradient display of the force field is entered with
the fragment copy as active entry and the map energy as
the only term.
Protein refinement, tests etc. [prp]
g: geometry display
f: forge structure
m: mend and modify
Incomplete side chains can be completed. Amino acid
types can be changed with automatic replacement of the
side chains.
w: add water molecules or isolated atoms
In a separate menu isolated atoms can be added to a
structure.
i: scan through isolated atoms
Isolated atoms can be scaned through and undesired ones
can be deleted.
p: scan through residues
The residues of a polymer can be scaned through. Tem
perature factors, real space correlation coefficients
and gradients with respect to the electron density map
may be shown for quality judgemnt.
o: optimize
r: refinement of set of ACTIVE entries
Refinement of full protein structures by energy minimi
zation. This option offers the possibility to apply
constraints in an automatic fashion (e.g. on all Calfs)
to guide the minimization in the desired direction. Use
of this option is generally indicated for structures
that have been built or modified on the Calf level. Upon
entering the user is asked which generic parts of the
residues he wants to keep fixed or constrained.
t: protein tests
Proteins can be tested for various geometric and ener
getic properties.
b: temperature (B-) factor display
Bonds are colored according in a half bond representa
tion such that the colors represent the temperature
factors of the adjacent atoms. Scale: yellow>40,
red>30, magenta>20, blue>10, else white.
s: define sets in a protein
Set definition by protein specific criteria is possible
here. The program generates a temporary Calf structure
on which the user can specify the residues that should
be affected. On these atom sets such as backbone, side
chaine etc. can be specified to belong to the set.
c: color residues
Residues can be colored according to users needs. Spec
ification is by type or name of residues.
z: search for SS bridge pattern
Single entry version of an option also provided in
menue xnr (see Section on
X-ray and NMR).
Mend and Modify Proteins
f: forge
s: complete single side chains
Upon picking an atom of a residue, any missing atom of
the side chain is inserted. Actually the option removes
all side-chain atoms and rebuilds the chain with the
chi angles that were available. Missing chi's are taken
from standard tables. All other geometry elements are
standard (bonds, valence angles).
a: complete all incomplete side chains
Same as option s, but applied to all incomplete side
chains.
c: change residue type
Upon picking an atom the user is asked to select the new
type of amino acid to be put in place of the old one.
Add Isolated Atoms (Water)
g: geometry display
f: forge
a: move atoms
To move an atom, different from the freshly inserted
one, this option can be used. It works exactly as the
corresponding option in the forge menu. As long as its
letter is highlighted, the insert option is silenced.
d: default naming
The program provides an automatic labelling of the
inserted atoms. The user can influence this by setting
this flag to 'u\q
c: change core number of atom to be inserted
Scan Isolated Atoms, Residues
g: geometry examination
a,s: select atom, residue
Select an isolated atom (residue) from a list. It will
be centered.
n: next
Proceeds to the next isolated atom (residue) and cen
ters it.
l: last
Returns to the last isolated atom (residue) and centers
it.
d: delete
The selected atom is deleted, and selection proceeds to
the next isolated atom. (not available for residues)
t: set thresholds
Without any thresholds set scanning proceeds according
to the natural sequence. When thresholds are set, all
residues which fulfil all thresholds are skipped. The
meaning of 'to fulfil\q depends on the quantity in ques
tion. RSCC values should lie above threshold, the val
ues of most other quantities below threshold. For
example, if one is only interested in a single quan
tity, the thresholds of all other quantities must be
set to their appropriate extreme (mostly maximum)
value.
p: print list of values (only for residues)
Refinement of Protein or DNA
f: forge structure (see
Forge)
o: optimize or run dynamics (see
MAB-force field)
s: modify set of stationary atoms
c: add positional constraints
In addition to the automatically assigned positional
constraints, individual specifications can be entered.
They are treated together in a common set.
w: set weights of constraints
For each set that has been subjected to positional
constraints a separate weight can be assigned. This may be
used, for example, to relax side chains and while keeping the
backbone fold unaffected by having constraints applied on
Calfs or backbone atoms.
Protein Tests [tst]
c: cis-peptides
A list of all peptide links that are in a cis conforma
tion is printed.
d: d-amino acids
Aist of all d-amino acids is printed.
b: beta chirality
Aist of all THR or ILE amino acids with non-natural
chirality at the beta carbon is printed.
s: find S-S bonds
Searches the picked structure for disulfide bonds. If
the geometry of two cysteins is compatible with a bond
the two sulfur atoms are highlighted and a bond is
introduced if it was missing before.
e: energy tests *
After evaluation the energy of the picked entry a menu
is offered, in which various energy-related quantities
can be displayed or printed.
o: write output to file
Printed output data can additionally be directed to a
file.
Energy tests for proteins [etst]
s: single residue energy
The internal energy of a picked residue is printed. For
a list of all single residue energies choose option l.
l: list of single residue energies
A list of the internal energies of all residues is
printed.
i: inter-residue interactions for a residue
The interaction energy of a picked residue with its
neighbors is printed. Energies are separated in attrac
tive and repulsive van der Waals and hydrogen bond con
tributions and listed separately for each residue with
which the picked one interacts
t: total of inter-residue interactions
The output of option i is given for all residues in the
structure.
w: highlight van der Waals conflicts
Each bond is highlighted, if its two end atoms together
experience van der Waals repulsions of more than 2
kcal/Mol.
e: graphical energy examination
The full energy examination option is entered as also
found in the
mab-menu.
o: write output to file
Printed output data can additionally be directed to a
file.