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.
Overlapping residues or molecules (for short entities) are occasionally observed in the PDB. Often these are cases like, for example, two sugars that bind equally well in the same active site, are both seen overlapping in the density, and are both entered in the PDB file as separate entities. This can cause some false positive error messsages further down the validation path, and therefore the second of the overlapping entities has been deleted before the validation continued. If you want to validate both situations, make it two PDB files, one for each sugar. And fudge reality a bit by making the occupancy of the sugar atoms 1.0 in both cases, because many validation options are not executed on atoms with low occupancy. If you go for this two-file option, please make sure that any side chains that have alternate locations depending on the sugar bound are selected in each of the two cases in agreement with the sugar that you keep for validation in that particular file.
307 ALA ( 307-) A -
Non-validating, descriptive output paragraph
Note: Ramachandran plot
In this Ramachandran plot x-signs represent glycines, squares represent
prolines, and plus-signs represent the other residues. If too many plus-
signs fall outside the contoured areas then the molecule is poorly refined
(or worse). Proline can only occur in the narrow region around phi=-60 that
also falls within the other contour islands.
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:
Crystal temperature (K) :298.000
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
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 near 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 restraints have been used in the refinement. This
can only be a problem for high resolution X-ray structures.
RMS Z-score for bond lengths: 0.289
RMS-deviation in bond distances: 0.007
Warning: Possible cell scaling problem
Comparison of bond distances with Engh and Huber [REF] standard values for
protein residues and Parkinson et al [REF] values for DNA/RNA shows a
significant systematic deviation. It could be that the unit cell used in
refinement was not accurate enough. The deformation matrix given below gives
the deviations found: the three numbers on the diagonal represent the
relative corrections needed along the A, B and C cell axis. These values are
1.000 in a normal case, but have significant deviations here (significant at
the 99.99 percent confidence level)
There are a number of different possible causes for the discrepancy. First the cell used in refinement can be different from the best cell calculated. Second, the value of the wavelength used for a synchrotron data set can be miscalibrated. Finally, the discrepancy can be caused by a dataset that has not been corrected for significant anisotropic thermal motion.
Please note that the proposed scale matrix has NOT been restrained to obey the space group symmetry. This is done on purpose. The distortions can give you an indication of the accuracy of the determination.
If you intend to use the result of this check to change the cell dimension of your crystal, please read the extensive literature on this topic first. This check depends on the wavelength, the cell dimensions, and on the standard bond lengths and bond angles used by your refinement software.
Unit Cell deformation matrix
| 0.998778 -0.000089 -0.000073| | -0.000089 0.998913 -0.000534| | -0.000073 -0.000534 0.998523|Proposed new scale matrix
| 0.018919 0.000002 0.000001| | 0.000001 0.013389 0.000007| | 0.000000 0.000004 0.007313|With corresponding cell
A = 52.857 B = 74.691 C = 136.747 Alpha= 90.061 Beta= 90.005 Gamma= 90.010
The CRYST1 cell dimensions
A = 52.920 B = 74.770 C = 136.940 Alpha= 90.000 Beta= 90.000 Gamma= 90.000
(Under-)estimated Z-score: 3.898
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.
2 TYR ( 2-) A -O -C N 111.26 -7.3 195 ARG ( 195-) A N CA C 98.35 -4.6 293 LEU ( 293-) A N CA C 98.77 -4.4
RMS Z-score for bond angles: 0.628
RMS-deviation in bond angles: 1.383
Error: Tau angle problems
The side chains of the residues listed in the table below contain a tau
angle (N-Calpha-C) that was found to deviate from te expected value by
more than 4.0 times the expected standard deviation. The number in the
table is the number of standard deviations this RMS value deviates from
the expected value.
195 ARG ( 195-) A 5.13 317 ALA ( 318-) A 4.75 293 LEU ( 293-) A 4.60 159 ASP ( 159-) A 4.59
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.
163 THR ( 163-) A -3.0 375 THR ( 376-) A -3.0 45 PRO ( 45-) A -2.4 340 SER ( 341-) A -2.2 56 ARG ( 56-) A -2.1 400 VAL ( 401-) A -2.0
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.
5 ASN ( 5-) A Poor phi/psi 18 GLU ( 18-) A Poor phi/psi 53 ASN ( 53-) A PRO omega poor 102 MET ( 102-) A Poor phi/psi 124 ARG ( 124-) A Poor phi/psi 129 VAL ( 129-) A PRO omega poor 163 THR ( 163-) A Poor phi/psi 268 LYS ( 268-) A Poor phi/psi 305 HIS ( 305-) A Poor phi/psi 349 ASN ( 350-) A Poor phi/psi 375 THR ( 376-) A Poor phi/psi 379 ASN ( 380-) A Poor phi/psi 380 ASP ( 381-) A Poor phi/psi 413 SER ( 414-) A Poor phi/psi 485 PRO ( 486-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -1.442
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!
5 ASN ( 5-) A 0 8 GLN ( 8-) A 0 12 SER ( 12-) A 0 17 PHE ( 17-) A 0 18 GLU ( 18-) A 0 19 TRP ( 19-) A 0 30 ARG ( 30-) A 0 31 TYR ( 31-) A 0 43 SER ( 43-) A 0 52 TYR ( 52-) A 0 53 ASN ( 53-) A 0 54 PRO ( 54-) A 0 55 PHE ( 55-) A 0 56 ARG ( 56-) A 0 58 TRP ( 58-) A 0 59 TRP ( 59-) A 0 62 TYR ( 62-) A 0 63 GLN ( 63-) A 0 64 PRO ( 64-) A 0 67 TYR ( 67-) A 0 69 LEU ( 69-) A 0 70 CYS ( 70-) A 0 73 SER ( 73-) A 0 75 ASN ( 75-) A 0 89 VAL ( 89-) A 0And so on for a total of 225 lines.
Standard deviation of omega values : 1.516
Warning: Backbone oxygen evaluation
The residues listed in the table below have an unusual backbone oxygen
For each of the residues in the structure, a search was performed to find 5-residue stretches in the WHAT IF database with superposable C-alpha coordinates, and some restraining on the neighbouring backbone oxygens.
In the following table the RMS distance between the backbone oxygen positions of these matching structures in the database and the position of the backbone oxygen atom in the current residue is given. If this number is larger than 1.5 a significant number of structures in the database show an alternative position for the backbone oxygen. If the number is larger than 2.0 most matching backbone fragments in the database have the peptide plane flipped. A manual check needs to be performed to assess whether the experimental data can support that alternative as well. The number in the last column is the number of database hits (maximum 80) used in the calculation. It is "normal" that some glycine residues show up in this list, but they are still worth checking!
144 GLY ( 144-) A 1.68 14 110 GLY ( 110-) A 1.64 17
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.
1 PCA ( 1-) A CG <-> 2 TYR ( 2-) A CE2 0.76 2.44 INTRA 1 PCA ( 1-) A CG <-> 2 TYR ( 2-) A CD2 0.39 2.81 INTRA 313 THR ( 314-) A CG2 <-> 314 PHE ( 315-) A N 0.30 2.70 INTRA 149 GLU ( 149-) A N <-> 156 GLN ( 156-) A OE1 0.26 2.44 INTRA 391 ARG ( 392-) A NH2 <-> 499 HOH ( 569 ) A O 0.25 2.45 INTRA 1 PCA ( 1-) A CG <-> 2 TYR ( 2-) A CZ 0.24 2.96 INTRA 305 HIS ( 305-) A NE2 <-> 355 ASP ( 356-) A OD2 0.22 2.48 INTRA BF 33 ALA ( 33-) A CB <-> 89 VAL ( 89-) A CG1 0.20 3.00 INTRA 391 ARG ( 392-) A NH2 <-> 499 HOH ( 603 ) A O 0.18 2.52 INTRA 14 VAL ( 14-) A CG2 <-> 37 PHE ( 37-) A CD2 0.16 3.04 INTRA BL 140 LYS ( 140-) A NZ <-> 171 GLU ( 171-) A CD 0.16 2.94 INTRA BF 1 PCA ( 1-) A CD <-> 2 TYR ( 2-) A CE2 0.15 3.05 INTRA 140 LYS ( 140-) A NZ <-> 171 GLU ( 171-) A OE1 0.13 2.57 INTRA BF 1 PCA ( 1-) A CD <-> 2 TYR ( 2-) A CD2 0.12 3.08 INTRA 321 LYS ( 322-) A NZ <-> 484 ASP ( 485-) A OD1 0.11 2.59 INTRA BL 124 ARG ( 124-) A NE <-> 138 ASP ( 138-) A OD2 0.09 2.61 INTRA 435 PHE ( 436-) A O <-> 478 ILE ( 479-) A N 0.09 2.61 INTRA 407 ASN ( 408-) A ND2 <-> 499 HOH ( 539 ) A O 0.09 2.61 INTRA 410 ASP ( 411-) A OD1 <-> 412 GLY ( 413-) A N 0.08 2.62 INTRA 134 TRP ( 134-) A O <-> 172 LYS ( 172-) A NZ 0.08 2.62 INTRA 305 HIS ( 305-) A CD2 <-> 355 ASP ( 356-) A OD2 0.08 2.72 INTRA BF 100 ASN ( 100-) A ND2 <-> 101 HIS ( 101-) A ND1 0.07 2.93 INTRA BL 374 ASP ( 375-) A C <-> 375 THR ( 376-) A CG2 0.07 3.03 INTRA 372 ASN ( 373-) A N <-> 376 THR ( 377-) A O 0.06 2.64 INTRA 146 GLY ( 146-) A N <-> 147 ASP ( 147-) A N 0.06 2.54 INTRA B3 2 TYR ( 2-) A N <-> 3 SER ( 3-) A N 0.05 2.55 INTRA B3 274 MET ( 274-) A N <-> 414 ASN ( 415-) A ND2 0.05 2.80 INTRA 465 LYS ( 466-) A NZ <-> 467 TYR ( 468-) A OH 0.03 2.67 INTRA 414 ASN ( 415-) A ND2 <-> 499 HOH ( 573 ) A O 0.03 2.67 INTRA 457 ILE ( 458-) A N <-> 460 ASN ( 461-) A O 0.03 2.67 INTRA 152 ASN ( 152-) A N <-> 153 ASP ( 153-) A N 0.03 2.57 INTRA BF 430 ASN ( 431-) A ND2 <-> 499 HOH ( 573 ) A O 0.01 2.69 INTRA 267 ARG ( 267-) A N <-> 268 LYS ( 268-) A N 0.01 2.59 INTRA B3 68 LYS ( 68-) A NZ <-> 117 SER ( 117-) A OG 0.01 2.69 INTRA 20 ARG ( 20-) A NH1 <-> 23 ASP ( 23-) A OD2 0.01 2.69 INTRA 170 LEU ( 170-) A CD1 <-> 202 MET ( 202-) A SD 0.01 3.39 INTRA BL 302 GLN ( 302-) A N <-> 303 ARG ( 303-) A N 0.01 2.59 INTRA B3
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.
72 ARG ( 72-) A -7.09 142 LYS ( 142-) A -6.12 2 TYR ( 2-) A -5.92 52 TYR ( 52-) A -5.91 8 GLN ( 8-) A -5.90 118 TYR ( 118-) A -5.57 342 ARG ( 343-) A -5.49 303 ARG ( 303-) A -5.42 284 TRP ( 284-) A -5.38 7 GLN ( 7-) A -5.36 279 ASN ( 279-) A -5.35 302 GLN ( 302-) A -5.32 237 LEU ( 237-) A -5.32 30 ARG ( 30-) A -5.31 88 ASN ( 88-) A -5.28 53 ASN ( 53-) A -5.18 348 GLN ( 349-) A -5.14 151 TYR ( 151-) A -5.09 134 TRP ( 134-) A -5.06
Chain identifier: A
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.
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.
15 HIS ( 15-) A 150 ASN ( 150-) A 414 ASN ( 415-) 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.
2 TYR ( 2-) A N 59 TRP ( 59-) A N 87 ASN ( 87-) A ND2 101 HIS ( 101-) A N 138 ASP ( 138-) A N 171 GLU ( 171-) A N 193 GLY ( 193-) A N 242 ILE ( 242-) A N 273 LYS ( 273-) A N 281 GLY ( 281-) A N 295 PHE ( 295-) A N 300 ASP ( 300-) A N 305 HIS ( 305-) A N 313 THR ( 314-) A N 315 TRP ( 316-) A N 315 TRP ( 316-) A NE1 336 ARG ( 337-) A NH2 343 TRP ( 344-) A N 354 ASN ( 355-) A ND2 356 TRP ( 357-) A N 433 TRP ( 434-) A N 465 LYS ( 466-) A NZ
The output gives the ion, the valency score for the ion itself, the valency score for the suggested alternative ion, and a series of possible comments *1 indicates that the suggested alternate atom type has been observed in the PDB file at another location in space. *2 indicates that WHAT IF thinks to have found this ion type in the crystallisation conditions as described in the REMARK 280 cards of the PDB file. *S Indicates that this ions is located at a special position (i.e. at a symmetry axis). N4 stands for NH4+.
497 CA ( 497-) A 0.76 1.00 Scores about as good as NA
188 ASP ( 188-) A H-bonding suggests Asn 246 ASP ( 246-) A H-bonding suggests Asn; but Alt-Rotamer
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 : -0.900 2nd generation packing quality : 0.104 Ramachandran plot appearance : -1.375 chi-1/chi-2 rotamer normality : -1.442 Backbone conformation : -0.884
Bond lengths : 0.289 (tight) Bond angles : 0.628 (tight) Omega angle restraints : 0.276 (tight) Side chain planarity : 0.236 (tight) Improper dihedral distribution : 0.603 B-factor distribution : 0.476 Inside/Outside distribution : 1.012
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, which have been calibrated against structures of similar resolution.
Resolution found in PDB file : 1.90
Structure Z-scores, positive is better than average:
1st generation packing quality : -0.4 2nd generation packing quality : -0.2 Ramachandran plot appearance : -1.2 chi-1/chi-2 rotamer normality : -0.8 Backbone conformation : -1.3
Bond lengths : 0.289 (tight) Bond angles : 0.628 (tight) Omega angle restraints : 0.276 (tight) Side chain planarity : 0.236 (tight) Improper dihedral distribution : 0.603 B-factor distribution : 0.476 Inside/Outside distribution : 1.012 ==============
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.