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.
209 BCT ( 301-) A -
Alternate atom indicators in PDB files are known to often be erroneous. It has been observed that alternate atom indicators are missing, or that there are too many of them. It is common to see that the distance between two atoms that should be covalently bound is far too big, but the distance between the alternate A of one of them and alternate B of the other is proper for a covalent bond. We have discovered many, many ways in which alternate atoms can be abused. The software tries to deal with most cases, but we know for sure that it cannot deal with all cases. If an alternate atom indicator problem is not properly solved, subsequent checks will list errors that are based on wrong coordinate combinations. So, any problem listed in this table should be solved before error messages further down in this report can be trusted.
205 SER ( 213-) A -
In case any of these residues shows up as poor or bad in checks further down this report, please check the consistency of the alternate atoms in this residue first, correct it yourself if needed, and run the validation again.
205 SER ( 213-) A - 210&HOH &HOH- & A& & 209&BCT & 301-& A& - & 1.0
The left-hand residue has been removed, and the right hand residue has been kept for validation. Be aware that WHAT IF calls everything a residue. Two residues are defined as overlapping if the two smallest ellipsoids encompassing the two residues interpenetrate by 33% of the longest axis. Many artefacts can actually cause this problem. The most often observed reason is alternative residue conformations expressed by two residues that accidentally both got 1.0 occupancy for all atoms.
210 HOH (HOH- ) A 209 BCT ( 301-) A - 1.0
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: B-factors outside the range 0.0 - 100.0
In principle, B-factors can have a very wide range of values, but in
practice, B-factors should not be zero while B-factors above 100.0
are a good indicator that the location of that atom is meaningless. Be
aware that the cutoff at 100.0 is arbitrary. 'High' indicates that atoms
with a B-factor > 100.0 were observed; 'Zero' indicates that atoms with
a B-factor of zero were observed.
14 GLU ( 22-) A High 80 GLU ( 88-) A High 81 GLU ( 89-) A High 83 GLU ( 91-) A High
Atoms want to move. That is the direct result of the second law of thermodynamics, in a somewhat weird way of thinking. Any way, many atoms seem to have more than one position where they like to sit, and they jump between them. The population difference between those sites (which is related to their energy differences) is seen in the occupancy factors. As also for atoms it is 'to be or not to be', these occupancies should add up to 1.0. Obviously, it is possible that they add up to a number less than 1.0, in cases where there are yet more, but undetected' rotamers/positions in play, but also in those cases a warning is in place as the information shown in the PDB file is less certain than it could have been. The residues listed below contain atoms that have an occupancy greater than zero, but all their alternates do not add up to one.
WARNING. Presently WHAT CHECK only deals with a maximum of two alternate positions. A small number of atoms in the PDB has three alternates. In those cases the warning given here should obviously be neglected! In a next release we will try to fix this.
76 GLU ( 84-) A 0.91
Obviously, the temperature at which the X-ray data was collected has some importance too:
Number of TLS groups mentione in PDB file header: 1
Crystal temperature (K) :293.000
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
Nomenclature related problems
Warning: Tyrosine convention problem
The tyrosine residues listed in the table below have their chi-2 not between
-90.0 and 90.0
97 TYR ( 105-) A 201 TYR ( 209-) A
59 PHE ( 67-) A
53 ASP ( 61-) A 121 ASP ( 129-) A 155 ASP ( 163-) A
14 GLU ( 22-) A 54 GLU ( 62-) A 86 GLU ( 94-) A 96 GLU ( 104-) A 170 GLU ( 178-) A
RMS Z-score for bond lengths: 0.539
RMS-deviation in bond distances: 0.013
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
| 0.997321 0.000699 -0.001055| | 0.000699 0.997402 -0.000597| | -0.001055 -0.000597 0.997039|Proposed new scale matrix
| 0.012008 -0.000008 0.000013| | -0.000008 0.012007 0.000007| | 0.000013 0.000007 0.012012|With corresponding cell
A = 83.277 B = 83.283 C = 83.253 Alpha= 90.069 Beta= 90.121 Gamma= 89.920
The CRYST1 cell dimensions
A = 83.498 B = 83.498 C = 83.498 Alpha= 90.000 Beta= 90.000 Gamma= 90.000
(Under-)estimated Z-score: 5.410
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.
145 ARG ( 153-) A CG CD NE 102.67 -4.6
14 GLU ( 22-) A 53 ASP ( 61-) A 54 GLU ( 62-) A 86 GLU ( 94-) A 96 GLU ( 104-) A 121 ASP ( 129-) A 155 ASP ( 163-) A 170 GLU ( 178-) A
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.
7 PRO ( 15-) 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.
3 ILE ( 11-) A omega poor 41 ASN ( 49-) A Poor phi/psi 47 MET ( 55-) A Poor phi/psi 56 MET ( 64-) A Poor phi/psi 63 ARG ( 71-) A Poor phi/psi 66 VAL ( 74-) A omega poor 67 GLN ( 75-) A omega poor 104 ASN ( 112-) A Poor phi/psi 110 GLN ( 118-) A Poor phi/psi 115 GLY ( 123-) A PRO omega poor 122 ASP ( 130-) A Poor phi/psi 128 GLN ( 136-) A Poor phi/psi 139 ASN ( 147-) A Poor phi/psi 145 ARG ( 153-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -0.312
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!
4 ARG ( 12-) A 0 8 VAL ( 16-) A 0 11 TRP ( 19-) A 0 14 GLU ( 22-) A 0 33 ILE ( 41-) A 0 35 GLU ( 43-) A 0 40 ALA ( 48-) A 0 41 ASN ( 49-) A 0 45 SER ( 53-) A 0 46 PRO ( 54-) A 0 47 MET ( 55-) A 0 52 SER ( 60-) A 0 53 ASP ( 61-) A 0 54 GLU ( 62-) A 0 56 MET ( 64-) A 0 62 ASP ( 70-) A 0 63 ARG ( 71-) A 0 67 GLN ( 75-) A 0 68 ASP ( 76-) A 0 73 HIS ( 81-) A 0 81 GLU ( 89-) A 0 98 ALA ( 106-) A 0 103 ASN ( 111-) A 0 108 ALA ( 116-) A 0 109 HIS ( 117-) A 0And so on for a total of 83 lines.
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!
126 GLY ( 134-) A 1.51 10
10 PRO ( 18-) A 0.19 LOW
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.
138 ASN ( 146-) A OD1 <-> 210 HOH ( 280 ) A O 0.20 2.20 INTRA BF 41 ASN ( 49-) A ND2 <-> 210 HOH ( 245 ) A O 0.07 2.63 INTRA 209 BCT ( 301-) A O1 <-> 210 HOH ( 282 ) A O 0.04 2.26 INTRA 135 LYS ( 143-) A A NZ <-> 210 HOH ( 278 ) A O 0.02 2.68 INTRA BF
Chain identifier: A
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.
81 GLU ( 89-) A -2.57 75 LEU ( 83-) 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.
41 ASN ( 49-) 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.
9 THR ( 17-) A OG1 67 GLN ( 75-) A NE2 78 ILE ( 86-) A N 123 THR ( 131-) A N 183 TYR ( 191-) A N 187 ASN ( 195-) A ND2 Only metal coordination for 73 HIS ( 81-) A ND1 Only metal coordination for 109 HIS ( 117-) A NE2 Only metal coordination for 114 HIS ( 122-) A NE2
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.
112 GLN ( 120-) A OE1 122 ASP ( 130-) A OD1
76 GLU ( 84-) A H-bonding suggests Gln
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 : 0.679 2nd generation packing quality : -0.903 Ramachandran plot appearance : 1.685 chi-1/chi-2 rotamer normality : -0.312 Backbone conformation : 0.388
Bond lengths : 0.539 (tight) Bond angles : 0.715 Omega angle restraints : 1.132 Side chain planarity : 0.838 Improper dihedral distribution : 0.832 B-factor distribution : 1.385 Inside/Outside distribution : 0.969
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 : 1.80
Structure Z-scores, positive is better than average:
1st generation packing quality : 1.0 2nd generation packing quality : -0.9 Ramachandran plot appearance : 1.8 chi-1/chi-2 rotamer normality : -0.1 Backbone conformation : 0.2
Bond lengths : 0.539 (tight) Bond angles : 0.715 Omega angle restraints : 1.132 Side chain planarity : 0.838 Improper dihedral distribution : 0.832 B-factor distribution : 1.385 Inside/Outside distribution : 0.969 ==============
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.