Please note that you are looking at an abridged version of the output (all checks that gave normal results have been removed from this report). You can have a look at the Full report instead.
In a colour picture, the residues that are part of a helix are shown in blue, strand residues in red. Preferred regions for helical residues are drawn in blue, for strand residues in red, and for all other residues in green. A full explanation of the Ramachandran plot together with a series of examples can be found at the WHAT_CHECK website.
Chain identifier: A
Coordinate problems, unexpected atoms, B-factor and occupancy checks
Warning: What type of B-factor?
WHAT IF does not yet know well how to cope with B-factors in case TLS has
been used. It simply assumes that the B-factor listed on the ATOM and HETATM
cards are the total B-factors. When TLS refinement is used that assumption
sometimes is not correct. TLS seems not mentioned in the header of the PDB
file. But anyway, if WHAT IF complains about your B-factors, and you think
that they are OK, then check for TLS related B-factor problems first.
Obviously, the temperature at which the X-ray data was collected has some importance too:
Temperature not mentioned in PDB file. This most likely means
that the temperature record is absent.
Room temperature assumed
Warning: Temperature factors given as "U", not as "B"
The average temperature factor found is very low. Probably they are given as
"U" values, and not as "B" values. Values will be multiplied by 8-pi-squared
for the analysis of B-factors.
Error: The B-factors of bonded atoms show signs of over-refinement
For each of the bond types in a protein a distribution was derived for the
difference between the square roots of the B-factors of the two atoms. All
bonds in the current protein were scored against these distributions. The
number given below is the RMS Z-score over the structure. For a structure
with completely restrained B-factors within residues, this value will be
around 0.35, for extremely high resolution structures refined with free
isotropic B-factors this number is expected to be near 1.0. Any value over
1.5 is sign of severe over-refinement of B-factors.
RMS Z-score : 2.394 over 381 bonds
Average difference in B over a bond : 9.38
RMS difference in B over a bond : 13.32
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
Warning: Directionality in bond lengths and no X-ray cell
Comparison of bond distances with Engh and Huber [REF] standard values for
protein residues and Parkinson et al [REF] standard values for DNA/RNA shows
a significant systematic deviation.
You have most probably seen symmetry problems earlier. Please correct these
and rerun this check to see the possible implications on the X-ray cell axes.
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.
1 PHE ( 249-) A CA CB CG 109.41 -4.4 28 HIS ( 276-) A CA CB CG 109.64 -4.2
Ramachandran Z-score : -6.706
Warning: Torsion angle evaluation shows unusual residues
The residues listed in the table below contain bad or abnormal
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.
37 THR ( 285-) A -2.8 13 GLU ( 261-) A -2.5 47 LEU ( 295-) A -2.5 15 THR ( 263-) A -2.4 36 ARG ( 284-) A -2.3 25 GLU ( 273-) A -2.2 30 ILE ( 278-) A -2.2 70 LEU ( 318-) A -2.2 4 ILE ( 252-) A -2.1 42 LEU ( 290-) A -2.1 33 LEU ( 281-) A -2.1
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.
13 GLU ( 261-) A Poor phi/psi 14 LEU ( 262-) A Poor phi/psi 18 SER ( 266-) A Poor phi/psi 26 ALA ( 274-) A Poor phi/psi 36 ARG ( 284-) A Poor phi/psi 47 LEU ( 295-) A Poor phi/psi 71 GLU ( 319-) A Poor phi/psi 73 TRP ( 321-) A PRO omega poor chi-1/chi-2 correlation Z-score : -7.177
chi-1/chi-2 correlation Z-score : -7.177
Warning: Unusual backbone conformations
For the residues listed in the table below, the backbone formed by itself and
two neighbouring 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 centre.
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!
13 GLU ( 261-) A 0 14 LEU ( 262-) A 0 25 GLU ( 273-) A 0 26 ALA ( 274-) A 0 27 ILE ( 275-) A 0 28 HIS ( 276-) A 0 29 TYR ( 277-) A 0 35 GLN ( 283-) A 0 36 ARG ( 284-) A 0 37 THR ( 285-) A 0 42 LEU ( 290-) A 0 44 THR ( 292-) A 0 46 ASN ( 294-) A 0 47 LEU ( 295-) A 0 64 LEU ( 312-) A 0 66 LEU ( 314-) A 0 68 MET ( 316-) A 0 71 GLU ( 319-) A 0 72 ASN ( 320-) A 0 73 TRP ( 321-) A 0 75 PRO ( 323-) A 0 76 ALA ( 324-) A 0 77 SER ( 325-) A 0 78 ILE ( 326-) A 0 79 ALA ( 327-) A 0 12 LEU ( 260-) A 1 38 GLU ( 286-) A 1 43 LYS ( 291-) A 1 65 SER ( 313-) A 1 69 ARG ( 317-) A 1 74 PRO ( 322-) A 1 7 ARG ( 255-) A 2 39 VAL ( 287-) A 2 62 ARG ( 310-) A 2
Backbone conformation Z-score : -2.191
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
restrained. This seems to be the case with the current structure too, as the
observed standard deviation is below 4.0 degrees.
Standard deviation of omega values : 0.504
Warning: Unusual PRO puckering phases
The proline residues listed in the table below have a puckering phase that is
not expected to occur in protein structures. Puckering parameters were
calculated by the method of Cremer and Pople [REF]. Normal PRO rings
approximately show a so-called envelope conformation with the C-gamma atom
above the plane of the ring (phi=+72 degrees), or a half-chair conformation
with C-gamma below and C-beta above the plane of the ring (phi=-90 degrees).
If phi deviates strongly from these values, this is indicative of a very
strange conformation for a PRO residue, and definitely requires a manual
check of the data. Be aware that this is a warning with a low confidence
level. See: Who checks the checkers? Four validation tools applied to eight
atomic resolution structures [REF].
8 PRO ( 256-) A -54.1 half-chair C-beta/C-alpha (-54 degrees) 45 PRO ( 293-) A -57.9 half-chair C-beta/C-alpha (-54 degrees) 74 PRO ( 322-) A -57.1 half-chair C-beta/C-alpha (-54 degrees)
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 centres of the two atoms. Although we believe that two water atoms at 2.4 A distance are too close, we only report water pairs that are closer than this rather short distance.
The last text-item on each line represents the status of the atom pair. If the final column contains the text 'HB', the bump criterion 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). BL indicates that the B-factors of the clashing atoms have a low B-factor thereby making this clash even more worrisome. INTRA and INTER indicate whether the clashes are between atoms in the same asymmetric unit, or atoms in symmetry related asymmetric units, respectively.
38 GLU ( 286-) A CD <-> 66 LEU ( 314-) A CD1 0.30 2.90 INTRA BF 28 HIS ( 276-) A NE2 <-> 29 TYR ( 277-) A CD2 0.27 2.83 INTRA BF 7 ARG ( 255-) A O <-> 30 ILE ( 278-) A CD1 0.26 2.54 INTRA BL 9 VAL ( 257-) A O <-> 19 ALA ( 267-) A CB 0.26 2.54 INTRA BL 4 ILE ( 252-) A CD1 <-> 5 LEU ( 253-) A N 0.25 2.75 INTRA BF 5 LEU ( 253-) A CD2 <-> 70 LEU ( 318-) A CD2 0.24 2.96 INTRA BF 15 THR ( 263-) A CB <-> 18 SER ( 266-) A OG 0.21 2.59 INTRA BF 16 VAL ( 264-) A O <-> 20 ASN ( 268-) A CB 0.20 2.60 INTRA BL 4 ILE ( 252-) A O <-> 30 ILE ( 278-) A CD1 0.19 2.61 INTRA BL 57 ASP ( 305-) A O <-> 61 SER ( 309-) A CB 0.18 2.62 INTRA BL 17 ARG ( 265-) A O <-> 21 CYS ( 269-) A CB 0.18 2.62 INTRA BL 2 ASP ( 250-) A O <-> 6 LEU ( 254-) A CD2 0.17 2.63 INTRA BL 9 VAL ( 257-) A CG2 <-> 10 ASP ( 258-) A N 0.17 2.83 INTRA BF 7 ARG ( 255-) A C <-> 30 ILE ( 278-) A CD1 0.16 3.04 INTRA BL 12 LEU ( 260-) A CD2 <-> 30 ILE ( 278-) A CG2 0.15 3.05 INTRA BF 54 GLU ( 302-) A O <-> 57 ASP ( 305-) A N 0.14 2.56 INTRA BL 67 GLY ( 315-) A O <-> 69 ARG ( 317-) A N 0.13 2.57 INTRA BL 8 PRO ( 256-) A CA <-> 28 HIS ( 276-) A O 0.13 2.67 INTRA BL 14 LEU ( 262-) A CD2 <-> 54 GLU ( 302-) A CG 0.11 3.09 INTRA BF 32 ASP ( 280-) A O <-> 35 GLN ( 283-) A N 0.11 2.59 INTRA BL 2 ASP ( 250-) A OD2 <-> 4 ILE ( 252-) A CG2 0.11 2.69 INTRA BF 8 PRO ( 256-) A O <-> 12 LEU ( 260-) A N 0.10 2.60 INTRA BL 47 LEU ( 295-) A CD1 <-> 48 GLY ( 296-) A N 0.09 2.91 INTRA BF 49 LYS ( 297-) A C <-> 51 SER ( 299-) A N 0.07 2.83 INTRA BL 7 ARG ( 255-) A O <-> 30 ILE ( 278-) A CG1 0.07 2.73 INTRA BL 27 ILE ( 275-) A CG2 <-> 33 LEU ( 281-) A N 0.07 3.03 INTRA BF 66 LEU ( 314-) A C <-> 68 MET ( 316-) A N 0.06 2.84 INTRA BL 2 ASP ( 250-) A O <-> 6 LEU ( 254-) A CG 0.06 2.74 INTRA BL 8 PRO ( 256-) A O <-> 9 VAL ( 257-) A C 0.06 2.54 INTRA BL 44 THR ( 292-) A CG2 <-> 46 ASN ( 294-) A N 0.05 3.05 INTRA BF 3 PRO ( 251-) A O <-> 4 ILE ( 252-) A C 0.05 2.55 INTRA BL 74 PRO ( 322-) A CB <-> 75 PRO ( 323-) A CD 0.04 3.06 INTRA BL 11 ASP ( 259-) A C <-> 13 GLU ( 261-) A N 0.04 2.86 INTRA BL 4 ILE ( 252-) A O <-> 30 ILE ( 278-) A CG1 0.04 2.76 INTRA BL 9 VAL ( 257-) A CG2 <-> 23 LYS ( 271-) A N 0.04 3.06 INTRA BF 29 TYR ( 277-) A C <-> 31 GLY ( 279-) A N 0.03 2.87 INTRA BL 7 ARG ( 255-) A CA <-> 8 PRO ( 256-) A CD 0.03 2.77 INTRA BL 54 GLU ( 302-) A O <-> 55 ILE ( 303-) A C 0.02 2.58 INTRA BL 8 PRO ( 256-) A O <-> 11 ASP ( 259-) A N 0.02 2.68 INTRA BL 2 ASP ( 250-) A CG <-> 4 ILE ( 252-) A CG2 0.02 3.18 INTRA BF 12 LEU ( 260-) A O <-> 13 GLU ( 261-) A CB 0.02 2.58 INTRA BL 73 TRP ( 321-) A CA <-> 74 PRO ( 322-) A CA 0.02 2.78 INTRA BL 5 LEU ( 253-) A O <-> 30 ILE ( 278-) A CG1 0.02 2.78 INTRA BL 32 ASP ( 280-) A O <-> 33 LEU ( 281-) A C 0.01 2.59 INTRA BL 27 ILE ( 275-) A CG1 <-> 36 ARG ( 284-) A NH1 0.01 3.09 INTRA BF 21 CYS ( 269-) A SG <-> 47 LEU ( 295-) A CD2 0.01 3.39 INTRA BF 74 PRO ( 322-) A CA <-> 75 PRO ( 323-) A CD 0.01 2.79 INTRA BL
Chain identifier: A
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.
69 ARG ( 317-) A -6.23 72 ASN ( 320-) A -5.01 46 ASN ( 294-) A -5.01
The protein is probably threaded correctly, but either poorly refined, or it is just a protein with an unusual (but correct) structure. The average packing score of 200 highly refined X-ray structures was -0.5+/-0.4 [REF].
Average for range 1 - 81 : -1.716
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
Chain identifier: A
Warning: Low packing Z-score for some residues
The residues listed in the table below have an unusual packing
environment according to the 2nd generation packing check. The score
listed in the table is a packing normality Z-score: positive means
better than average, negative means worse than average. Only residues
scoring less than -2.50 are listed here. These are the unusual
residues in the structure, so it will be interesting to take a
special look at them.
47 LEU ( 295-) A -2.53
Chain identifier: A
Water, ion, and hydrogenbond related checks
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 favourable 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. Be aware,
though, that if the topology could not be determined for one or more
ligands, then this option will make errors.
28 HIS ( 276-) A 35 GLN ( 283-) A
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.
Waters are not listed by this option.
10 ASP ( 258-) A N 37 THR ( 285-) A N 51 SER ( 299-) A N 62 ARG ( 310-) A NE 65 SER ( 313-) A N 70 LEU ( 318-) A N 78 ILE ( 326-) A N
Side-chain hydrogen bond acceptors buried inside the protein normally form hydrogen bonds within the protein. If there are any not hydrogen bonded in the optimized hydrogen bond network they will be listed here.
Waters are not listed by this option.
38 GLU ( 286-) A OE2
54 GLU ( 302-) A H-bonding suggests Gln 57 ASP ( 305-) A H-bonding suggests Asn
The second part of the table mostly gives an impression of how well the model conforms to common refinement restraint values. The first part of the table shows a number of global quality indicators.
Structure Z-scores, positive is better than average:
1st generation packing quality : -3.041 2nd generation packing quality : -2.013 Ramachandran plot appearance : -6.706 (bad) chi-1/chi-2 rotamer normality : -7.177 (bad) Backbone conformation : -2.191
Bond lengths : 0.938 Bond angles : 0.966 Omega angle restraints : 0.092 (tight) Side chain planarity : 0.092 (tight) Improper dihedral distribution : 0.783 B-factor distribution : 2.394 (loose) Inside/Outside distribution : 0.927 ==============
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). (see also http://swift.cmbi.ru.nl/gv/whatcheck for a course and extra inform 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). Ion Checks I.D.Brown and K.K.Wu, Empirical Parameters for Calculating Cation-Oxygen Bond Valences Acta Cryst. B32, 1957--1959 (1975). M.Nayal and E.Di Cera, Valence Screening of Water in Protein Crystals Reveals Potential Na+ Binding Sites J.Mol.Biol. 256 228--234 (1996). P.Mueller, S.Koepke and G.M.Sheldrick, Is the bond-valence method able to identify metal atoms in protein structures? Acta Cryst. D 59 32--37 (2003). Checking checks K.Wilson, C.Sander, R.W.W.Hooft, G.Vriend, et al. Who checks the checkers J.Mol.Biol. (1998) 276,417-436.