WHATIF is a suite of programs for the graphical display, manipulation and analysis of proteins. In the context of this Special Section the utility program FULCHK will be used to check the stereochemistry of the structure and the syntax of the PDB file.
The best way to see what FULCHK will do for you is to browse the output from an analysis of the pyrococcus woesei TATA-binding protein as shown below.
************************************************************************
********** REPORT OF PROTEIN ANALYSIS by the WHAT IF program **********
************************************************************************
Date : 1998-10-21
This report was created by WHAT IF version 19970813-1517
INTRODUCTION
------------
This document contains a report of findings by the WHAT IF program
during the analysis of one or more proteins. It contains a separate section
for each of the proteins that have been analysed. Each reported fact has
an assigned severity, one of:
* error: severe errors encountered during the analyses. Items marked
as errors are considered severe problems requiring immediate
attention.
* warning: Either less severe problems or uncommon structural
features. These still need special attention.
* note: Statistical values, plots, or other verbose results of
tests and analyses that have been performed.
If alternate conformations are present, only the first is
evaluated.
Hydrogen atoms are only included if explicitly requested, and even then
they are not used by all checks.
Legend
------
Some notations need a little explanation:
RESIDUE: Residues in tables are normally given in 3-5 parts:
- A number. This is the internal sequence number of the residue used
by WHAT IF.
- The residue name. Normally this is a three letter amino acid name.
- The sequence number, between brackets. This is the residue number
as it was given in the input file. It can be followed by the insertion
code.
- The chain identifier. A single character. If no chain identifier
was given in the input file, this will be invisible.
- A model number (only for NMR structures).
Z-VALUE: To indicate the normality of a score, the score may be
expressed as a Z-value or Z-score. This is just the number of
standard deviations that the score deviates from the expected
value. A property of Z-values is that the root-mean-square of a
group of Z-values (the RMS Z-value) is expected to be 1.0. Z-values
above 4.0 and below -4.0 are very uncommon. If a Z-score is used in
WHAT IF, the accompanying text will explain how the expected value
and standard deviation were obtained.
========================================================================
==== Compound code ../pwtbp_mnra.pdb ====
========================================================================
# 1 # Error: Missing unit cell information
No SCALE matrix is given in the PDB file.
# 2 # Error: Missing symmetry information
Problem: No CRYST1 card is given in the PDB file.
# 3 # Note: No rounded coordinates detected
No significant rounding of atom coordinates has been detected.
# 4 # Note: Valine nomenclature OK
No errors were detected in valine nomenclature.
# 5 # Note: Threonine nomenclature OK
No errors were detected in threonine nomenclature.
# 6 # Note: Isoleucine nomenclature OK
No errors were detected in isoleucine nomenclature.
# 7 # Note: Leucine nomenclature OK
No errors were detected in leucine nomenclature.
# 8 # Note: Arginine nomenclature OK
No errors were detected in arginine nomenclature.
# 9 # Note: Tyrosine torsion conventions OK
No errors were detected in tyrosine torsion angle conventions.
# 10 # Warning: Phenylalanine convention problem
The phenylalanine residues listed in the table below have their
chi-2 not between -90.0 and 90.0.
56 PHE ( 60 )
130 PHE ( 134 )
147 PHE ( 151 )
# 11 # Warning: Aspartic acid convention problem
The aspartic acid residues listed in the table below have their
chi-2 not between -90.0 and 90.0, or their proton on OD1 instead of
OD2.
15 ASP ( 19 )
21 ASP ( 25 )
47 ASP ( 51 )
48 ASP ( 52 )
97 ASP ( 101 )
114 ASP ( 118 )
162 ASP ( 166 )
175 ASP ( 179 )
# 12 # Warning: Glutamic acid convention problem
The glutamic acid residues listed in the table below have their
chi-3 outside the -90.0 to 90.0 range, or their proton on OE1 instead
of OE2.
23 GLU ( 27 )
38 GLU ( 42 )
110 GLU ( 114 )
126 GLU ( 130 )
165 GLU ( 169 )
# 13 # Note: Heavy atom naming OK
No errors were detected in the atom names for non-hydrogen atoms.
# 14 # Note: Chirality OK
All protein atoms have proper chirality.
# 15 # Note: Improper dihedral angle distribution OK
The RMS Z-score for all improper dihedrals in the structure is within
normal ranges.
Improper dihedral RMS Z-score : 0.730
# 16 # Note: Chain names are OK
All chain names assigned to polymer molecules are unique, and all
residue numbers are strictly increasing within each chain.
# 17 # Note: Weights checked OK
All atomic occupancy factors ('weights') fall in the 0.0--1.0 range.
# 18 # Note: No missing atoms detected
All expected atoms are present.
# 19 # Warning: C-terminal oxygen atoms missing
The C-atoms listed in the table below belong to a C-terminal residue
in a protein chain, but the C-terminal oxygen ("O2" or "OXT") that it
should be bound to was not found.
180 LEU ( 184 ) C
# 20 # Note: No extra C-terminal groups found
No C-terminal groups are present for non C-terminal residues
# 21 # Note: All bond lengths OK
All bond lengths are in agreement with standard bond lengths using
a tolerance of 4 sigma (both standard values and sigma for amino
acid residues have been taken from Engh and Huber [REF], for
DNA/RNA from Parkinson et al [REF])
# 22 # Warning: Low bond length variability
Bond lengths were found to deviate less than normal from the mean
Engh and Huber [REF] and/or Parkinson et al [REF] standard bond
lengths. The RMS Z-score given below is expected to be around 1.0
for a normally restrained data set. The fact that it is lower than
0.667 in this structure might indicate that too-strong constraints
have been used in the refinement. This can only be a problem
for high resolution X-ray structures.
RMS Z-score for bond lengths: 0.371
RMS-deviation in bond distances: 0.008
# 23 # Note: No bond length directionality
Comparison of bond distances with Engh and Huber [REF] standard
values for protein residues and Parkinson et al [REF] values for
DNA/RNA does not show significant systematic deviations.
# 24 # Warning: Unusual bond angles
The bond angles listed in the table below were found to deviate
more than 4 sigma from standard bond angles (both standard values
and sigma for protein residues have been taken from Engh and Huber
[REF], for DNA/RNA from Parkinson et al [REF]). In the table below
for each strange angle the bond angle and the number of standard
deviations it differs from the standard values is given. Please
note that disulphide bridges are neglected. Atoms starting with "<"
belong to the previous residue in the sequence.
54 LEU ( 58 ) N CA C 99.922 -4.0
104 SER ( 108 ) N CA C 99.669 -4.1
112 ASN ( 116 ) N CA C 98.712 -4.5
# 25 # Warning: Low bond angle variability
Bond angles were found to deviate less than normal from the
standard bond angles (normal values for protein residues were taken
from Engh and Huber [REF], for DNA/RNA from Parkinson et al
[REF]). The RMS Z-score given below is expected to be around 1.0
for a normally restrained data set. More common values are around
1.55. The fact that it is lower than 0.667 in this structure might
indicate that too-strong constraints have been used in the
refinement. This can only be a problem for high resolution X-ray
structures.
RMS Z-score for bond angles: 0.659
RMS-deviation in bond angles: 1.587
# 26 # Note: Side chain planarity OK
All of the side chains of residues that have a planar group are
planar within expected RMS deviations.
# 27 # Note: Atoms connected to aromatic rings OK
All of the atoms that are connected to planar aromatic rings in side
chains of amino-acid residues are in the plane within expected RMS
deviations.
# 28 # Note: PRO puckering amplitude OK
Puckering amplitudes for all PRO residues are within normal ranges.
# 29 # Note: PRO puckering phases OK
Puckering phases for all PRO residues are normal
# 30 # Warning: Torsion angle evaluation shows unusual residues
The residues listed in the table below contain bad or abnormal
torsion angles.
These scores give an impression of how ``normal'' the torsion
angles in protein residues are. All torsion angles except omega are
used for calculating a `normality' score. Average values and
standard deviations were obtained from the residues in the WHAT IF
database. These are used to calculate Z-scores. A residue with a
Z-score of below -2.0 is poor, and a score of less than -3.0 is
worrying. For such residues more than one torsion angle is in a
highly unlikely position.
127 PRO ( 131 ) -2.7850
61 LEU ( 65 ) -2.6740
86 ILE ( 90 ) -2.3130
107 ILE ( 111 ) -2.3019
51 VAL ( 55 ) -2.1532
5 LEU ( 9 ) -2.1097
139 GLU ( 143 ) -2.0070
# 31 # Warning: Backbone torsion angle evaluation shows unusual conformations
The residues listed in the table below have abnormal backbone torsion
angles.
Residues with ``forbidden'' phi-psi combinations are listed, as
well as residues with unusual omega angles (deviating by more than
3 sigma from the normal value). Please note that it is normal if
about 5 percent of the residues is listed here as having unusual
phi-psi combinations.
17 PHE ( 21 ) Poor phi/psi
31 ASN ( 35 ) Poor phi/psi
48 ASP ( 52 ) PRO omega poor
127 PRO ( 131 ) Poor PRO-phi
139 GLU ( 143 ) PRO omega poor
# 32 # Note: Ramachandran Z-score OK
The score expressing how well the backbone conformations of all residues
are corresponding to the known allowed areas in the Ramachandran plot is
within expected ranges for well-refined structures.
Ramachandran Z-score : -0.757
# 33 # Warning: Omega angles too tightly restrained
The omega angles for trans-peptide bonds in a structure are
expected to give a gaussian distribution with the average around
+178 degrees and a standard deviation around 5.5 degrees. These
expected values were obtained from very accurately determined
structures. Many protein structures are too tightly constrained.
This seems to be the case with the current structure, as the
observed standard deviation is below 4.0 degrees.
Standard deviation of omega values : 1.361
# 34 # Note: chi-1/chi-2 angle correlation Z-score OK
The score expressing how well the chi-1/chi-2 angles of all residues
are corresponding to the populated areas in the database is
within expected ranges for well-refined structures.
chi-1/chi-2 correlation Z-score : -1.237
# 35 # Note: Ramachandran plot
In this Ramachandran plot X-signs represent glycines, squares represent
prolines and small plus-signs represent the other residues. If too many
plus-signs fall outside the contoured areas then the molecule is poorly
refined (or worse).
In a colour picture, the residues that are part of a helix are
shown in blue, strand residues in red. "Allowed" regions for
helical residues are drawn in blue, for strand residues in red, and
for all other residues in green.
In the TeX file, a plot has been inserted here
Chain without chain identifier
# 36 # Note: Inside/Outside residue distribution normal
The distribution of residue types over the inside and the outside of the
protein is normal.
inside/outside RMS Z-score : 0.881
# 37 # Note: Inside/Outside RMS Z-score plot
The Inside/Outside distribution normality RMS Z-score over a 15
residue window is plotted as function of the residue number. High
areas in the plot (above 1.5) indicate unusual inside/outside
patterns.
In the TeX file, a plot has been inserted here
Chain without chain identifier
# 38 # Note: Secondary structure
This is the secondary structure according to DSSP. Only helix (H), strand
(S), turn (T) and coil (blank) are shown. [REF]
Secondary structure assignment
The DSSP executable was not found
/srv/local/whatif/19970813.sgi/dssp/DSSP.EXE
WARNING. You don't have the DSSP program installed. Therefore
the emulator will be used. This emulator gives rather poor results,
but it prevents WHAT IF from crashing. See the writeup about this.
10 20 30 40 50 60
| | | | | |
1 - 60 SKVKLRIENIVASVDLFAQLDLEKVLDLCPNSKYNPEEFPGIICHLDDPKVALLIFSSGK
1 - 60 SSS SSSSSSS T TH 3HH33T TT T 3T SSSSS SSSSST T
70 80 90 100 110 120
| | | | | |
61 - 120 LVVTGAKSVQDIERAVAKLAQKLKSIGVKFKRAPQIDVQNMVFSGDIGREFNLDVVALTL
61 - 120 SSSSTS THHHHHHHHHHHHHHHHHHT SSS SSSSSSS T THHHHHHHT
130 140 150 160 170 180
| | | | | |
121 - 180 PNCEYEPEQFPGVIYRVKEPKSVILLFSSGKIVCSGAKSEADAWEAVRKLLRELDKYGLL
121 - 180 T S T 3T SSSSS SSSSST T SSSST THHHHHHHHHHHHHHHHHTT
# 39 # Error: Abnormally short interatomic distances
The pairs of atoms listed in the table below have an unusually
short distance.
The contact distances of all atom pairs have been checked. Two
atoms are said to `bump' if they are closer than the sum of their
Van der Waals radii minus 0.40 Angstrom. For hydrogen bonded pairs
a tolerance of 0.55 Angstrom is used. The first number in the
table tells you how much shorter that specific contact is than the
acceptable limit. The second distance is the distance between the
centers of the two atoms.
The last text-item on each line represents the status of the atom
pair. The text `INTRA' means that the bump is between atoms that
are explicitly listed in the PDB file. `INTER' means it is an
inter-symmetry bump. If the final column contains the text 'HB',
the bump criterium was relaxed because there could be a hydrogen
bond. Similarly relaxed criteria are used for 1--3 and 1--4
interactions (listed as 'B2' and 'B3', respectively). If the last
column is 'BF', the sum of the B-factors of the atoms is higher
than 80, which makes the appearance of the bump somewhat less
severe because the atoms probably aren't there anyway.
Bumps between atoms for which the sum of their occupancies is lower
than one are not reported. In any case, each bump is listed in only
one direction.
51 VAL ( 55 ) CG1 -- 52 ALA ( 56 ) N 0.185 2.915 INTRA
100 ASN ( 104 ) ND2 -- 156 GLY ( 160 ) CA 0.159 2.941 INTRA
100 ASN ( 104 ) ND2 -- 156 GLY ( 160 ) N 0.122 2.878 INTRA
121 PRO ( 125 ) O -- 122 ASN ( 126 ) C 0.059 2.741 INTRA BF
14 VAL ( 18 ) CG1 -- 15 ASP ( 19 ) N 0.034 3.066 INTRA BF
150 GLY ( 154 ) N -- 151 LYS ( 155 ) N 0.032 2.568 INTRA B3
9 ASN ( 13 ) CG -- 10 ILE ( 14 ) N 0.026 3.074 INTRA
86 ILE ( 90 ) N -- 87 GLY ( 91 ) N 0.026 2.574 INTRA BF
126 GLU ( 130 ) CA -- 127 PRO ( 131 ) CD 0.025 2.775 INTRA B3
115 VAL ( 119 ) O -- 116 VAL ( 120 ) C 0.021 2.779 INTRA
59 GLY ( 63 ) N -- 60 LYS ( 64 ) N 0.019 2.581 INTRA B3
25 VAL ( 29 ) O -- 26 LEU ( 30 ) C 0.012 2.788 INTRA BF
24 LYS ( 28 ) O -- 25 VAL ( 29 ) C 0.003 2.797 INTRA BF
11 VAL ( 15 ) O -- 99 GLN ( 103 ) N 0.002 2.548 INTRA HB
# 40 # Warning: Abnormal packing environment for some residues
The residues listed in the table below have an unusual packing
environment.
The packing environment of the residues is compared with the
average packing environment for all residues of the same type in
good PDB files. A low packing score can indicate one of several
things: Poor packing, misthreading of the sequence through the
density, crystal contacts, contacts with a co-factor, or the
residue is part of the active site. It is not uncommon to see a few
of these, but in any case this requires further inspection of the
residue.
129 GLN ( 133 ) -5.72
92 ARG ( 96 ) -5.40
38 GLU ( 42 ) -5.08
# 41 # Note: No series of residues with bad packing environment
There are no stretches of three or more residues each having a quality
control score worse than -4.0.
# 42 # Note: Structural average packing environment OK
The structural average quality control value is within normal ranges.
Average for range 1 - 180 : 0.211
# 43 # Note: Quality value plot
The quality value smoothed over a 10 residue window is plotted as
function of the residue number. Low areas in the plot (below
-2.0) indicate "unusual" packing.
In the TeX file, a plot has been inserted here
Chain without chain identifier
# 44 # Note: Second generation packing environment OK
None of the individual amino acid residues has a bad packing environment.
# 45 # Note: No series of residues with abnormal new packing environment
There are no stretches of four or more residues each having a quality
control Z-score worse than -1.75.
# 46 # Note: Structural average packing Z-score OK
The structural average for the second generation quality control
value is within normal ranges.
All contacts : Average = 0.353 Z-score = 2.49
BB-BB contacts : Average = 0.672 Z-score = 4.82
BB-SC contacts : Average = -0.152 Z-score = -0.74
SC-BB contacts : Average = 0.425 Z-score = 2.76
SC-SC contacts : Average = -0.157 Z-score = -0.55
# 47 # Note: Second generation quality Z-score plot
The second generation quality Z-score smoothed over a 10 residue window
is plotted as function of the residue number. Low areas in the plot (below
-1.3) indicate "unusual" packing.
In the TeX file, a plot has been inserted here
Chain without chain identifier
# 48 # Note: Backbone oxygen evaluation OK
All residues for which the local backbone conformation could be
found in the WHAT IF database have a normal backbone oxygen
position.
# 49 # Note: Rotamers checked OK
None of the residues that have a normal backbone environment have
abnormal rotamers.
# 50 # Warning: Unusual backbone conformations
For the residues listed in the table below, the backbone formed by
itself and two neighboring residues on either side is in a
conformation that is not seen very often in the database of solved
protein structures. The number given in the table is the number of
similar backbone conformations in the database with the same amino
acid in the center.
For this check, backbone conformations are compared with database
structures using C-alpha superpositions with some restraints on the
backbone oxygen positions.
A residue mentioned in the table can be part of a strange loop, or
there might be something wrong with it or its directly surrounding
residues. There are a few of these in every protein, but in any
case it is worth looking at!
139 GLU ( 143 ) 0
48 ASP ( 52 ) 1
17 PHE ( 21 ) 2
50 LYS ( 54 ) 2
107 ILE ( 111 ) 2
141 LYS ( 145 ) 2
# 51 # Note: Backbone conformation Z-score OK
The backbone conformation analysis gives a score that is normal
for well refined protein structures.
Backbone conformation Z-score : 0.445
# 52 # Warning: Average B-factor problem
The average B-factor for all buried protein atoms normally lies between
10--20. Values around 3--5 are expected for X-ray studies performed
at liquid nitrogen temperature.
Because of the extreme value for the average B-factor, no further analysis
of the B-factors is performed.
Average B-factor for buried atoms : 38.561
# 53 # Note: B-factor plot
The average atomic B-factor per residue is plotted as function of
the residue number.
In the TeX file, a plot has been inserted here
Chain without chain identifier
# 54 # Error: HIS, ASN, GLN side chain flips
Listed here are Histidine, Asparagine or Glutamine residues for
which the orientation determined from hydrogen bonding analysis are
different from the assignment given in the input. Either they could
form energetically more favorable hydrogen bonds if the terminal
group was rotated by 180 degrees, or there is no assignment in the
input file (atom type 'A') but an assignment could be made. If a
residue is marked ``flexible'' the flipped conformation is only
slightly better than the non-flipped conformation.
100 ASN ( 104 )
# 55 # Note: Histidine type assignments
For all complete HIS residues in the structure a tentative
assignment to HIS-D (protonated on ND1), HIS-E (protonated on NE2),
or HIS-H (protonated on both ND1 and NE2, positively charged) is
made based on the hydrogen bond network. A second assignment is
made based on which of the Engh and Huber [REF] histidine
geometries fits best to the structure.
In the table below all normal histidine residues are listed. The
assignment based on the geometry of the residue is listed first,
together with the RMS Z-score for the fit to the Engh and Huber
parameters. For all residues where the H-bond assignment is
different, the assignment is listed in the last columns, together
with its RMS Z-score to the Engh and Huber parameters.
As always, the RMS Z-scores should be close to 1.0 if the residues
were restrained to the Engh and Huber parameters during refinement.
Please note that because the differences between the geometries of
the different types are small it is possible that the geometric
assignment given here does not correspond to the type used in
refinement. This is especially true if the RMS Z-scores are much
higher than 1.0.
If the two assignments differ, or the ``geometry'' RMS Z-score is high,
it is advisable to verify the hydrogen bond assignment, check the
HIS type used during the refinement and possibly adjust it.
45 HIS ( 49 ) HIS-H 0.13 HIS-E 0.61
# 56 # Warning: Buried unsatisfied hydrogen bond donors
The buried hydrogen bond donors listed in the table below have a
hydrogen atom that is not involved in a hydrogen bond in the
optimized hydrogen bond network.
Hydrogen bond donors that are buried inside the protein normally
use all of their hydrogens to form hydrogen bonds within the
protein. If there are any non hydrogen bonded buried hydrogen bond
donors in the structure they will be listed here. In very good
structures the number of listed atoms will tend to zero.
3 VAL ( 7 ) N
41 GLY ( 45 ) N
45 HIS ( 49 ) N
48 ASP ( 52 ) N
68 SER ( 72 ) N
92 ARG ( 96 ) N
109 ARG ( 113 ) N
136 ARG ( 140 ) N
139 GLU ( 143 ) N
157 ALA ( 161 ) N
159 SER ( 163 ) N
# 57 # Note: Buried hydrogen bond acceptors OK
All buried polar side-chain hydrogen bond acceptors are involved in a
hydrogen bond in the optimized hydrogen bond network.
# 58 # Note: Summary report for users of a structure
This is an overall summary of the quality of the structure as
compared with current reliable structures. This summary is most
useful for biologists seeking a good structure to use for modelling
calculations.
The second part of the table mostly gives an impression of how well
the model conforms to common refinement constraint values. The
first part of the table shows a number of constraint-independent
quality indicators.
Structure Z-scores, positive is better than average:
1st generation packing quality : 1.779
2nd generation packing quality : 2.486
Ramachandran plot appearance : -0.757
chi-1/chi-2 rotamer normality : -1.237
Backbone conformation : 0.445
RMS Z-scores, should be close to 1.0:
Bond lengths : 0.371 (tight)
Bond angles : 0.659 (tight)
Omega angle restraints : 0.247 (tight)
Side chain planarity : 0.306 (tight)
Improper dihedral distribution : 0.730
Inside/Outside distribution : 0.881
REFERENCES
==========
WHAT IF
G.Vriend,
WHAT IF: a molecular modelling and drug design program,
J. Mol. Graph. 8, 52--56 (1990).
WHAT_CHECK (verification routines from WHAT IF)
R.W.W.Hooft, G.Vriend, C.Sander and E.E.Abola,
Errors in protein structures
Nature 381, 272 (1996).
Bond lengths and angles, protein residues
R.Engh and R.Huber,
Accurate bond and angle parameters for X-ray protein structure
refinement,
Acta Crystallogr. A47, 392--400 (1991).
Bond lengths and angles, DNA/RNA
G.Parkinson, J.Voitechovsky, L.Clowney, A.T.Bruenger and H.Berman,
New parameters for the refinement of nucleic acid-containing structures
Acta Crystallogr. D52, 57--64 (1996).
DSSP
W.Kabsch and C.Sander,
Dictionary of protein secondary structure: pattern
recognition of hydrogen bond and geometrical features
Biopolymers 22, 2577--2637 (1983).
Hydrogen bond networks
R.W.W.Hooft, C.Sander and G.Vriend,
Positioning hydrogen atoms by optimizing hydrogen bond networks in
protein structures
PROTEINS, 26, 363--376 (1996).
Matthews' Coefficient
B.W.Matthews
Solvent content of Protein Crystals
J. Mol. Biol. 33, 491--497 (1968).
Protein side chain planarity
R.W.W. Hooft, C. Sander and G. Vriend,
Verification of protein structures: side-chain planarity
J. Appl. Cryst. 29, 714--716 (1996).
Puckering parameters
D.Cremer and J.A.Pople,
A general definition of ring puckering coordinates
J. Am. Chem. Soc. 97, 1354--1358 (1975).
Quality Control
G.Vriend and C.Sander,
Quality control of protein models: directional atomic
contact analysis,
J. Appl. Cryst. 26, 47--60 (1993).
Ramachandran plot
G.N.Ramachandran, C.Ramakrishnan and V.Sasisekharan,
Stereochemistry of Polypeptide Chain Conformations
J. Mol. Biol. 7, 95--99 (1963).
Symmetry Checks
R.W.W.Hooft, C.Sander and G.Vriend,
Reconstruction of symmetry related molecules from protein
data bank (PDB) files
J. Appl. Cryst. 27, 1006--1009 (1994).
Back to Post-structure Analysis
Revised: Wednesday, 21-Oct-1998 13:45:36 EDT