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.
Plausible side chain atoms were detected with (near) zero occupancy
When crystallographers do not see an atom they either leave it out completely, or give it an occupancy of zero or a very high B-factor. WHAT IF neglects these atoms. In this case some atoms were found with zero occupancy, but with coordinates that place them at a plausible position. Although WHAT IF knows how to deal with missing side chain atoms, validation will go more reliable if all atoms are presnt. So, please consider manually setting the occupancy of the listed atoms at 1.0.
1 HIS ( 4-) A - CG 1 HIS ( 4-) A - ND1 1 HIS ( 4-) A - CD2 1 HIS ( 4-) A - CE1 1 HIS ( 4-) A - NE2 257 LYS ( 261-) A - CB 257 LYS ( 261-) A - CG 257 LYS ( 261-) A - CD 257 LYS ( 261-) A - CE 257 LYS ( 261-) A - NZ
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: Occupancies atoms do not add up to 1.0.
In principle, the occupancy of all alternates of one atom should add up till
1.0. A valid exception is the missing atom (i.e. an atom not seen in the
electron density) that is allowed to have a 0.0 occupancy. Sometimes this
even happens when there are no alternate atoms given...
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.
61 HIS ( 64-) A 0.62
Obviously, the temperature at which the X-ray data was collected has some importance too:
Temperature cannot be read from the PDB file. This most likely means that
the temperature is listed as NULL (meaning unknown) in the PDB file.
Warning: More than 2 percent of buried atoms has low B-factor
For protein structures determined at room temperature, no more than
about 1 percent of the B factors of buried atoms is below 5.0.
Percentage of buried atoms with B less than 5 : 3.19
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 : 1.505 over 1853 bonds
Average difference in B over a bond : 2.93
RMS difference in B over a bond : 3.81
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
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.
64 ASN ( 67-) A C CA CB 102.48 -4.0 204 THR ( 208-) A N CA C 95.20 -5.7 257 LYS ( 261-) A N CA C 98.87 -4.4
204 THR ( 208-) A 6.01 257 LYS ( 261-) A 4.79 124 TYR ( 128-) A 4.26
Tau angle RMS Z-score : 1.678
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.
39 PRO ( 42-) A -2.6 47 SER ( 50-) A -2.3 163 ILE ( 167-) A -2.2 147 GLY ( 151-) A -2.1 89 GLN ( 92-) 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.
26 SER ( 29-) A PRO omega poor 61 HIS ( 64-) A Poor phi/psi 108 LYS ( 111-) A Poor phi/psi 197 PRO ( 201-) A PRO omega poor 239 ASP ( 243-) A Poor phi/psi 248 LYS ( 252-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -2.753
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 TYR ( 7-) A 0 7 HIS ( 10-) A 0 16 ASP ( 19-) A 0 17 PHE ( 20-) A 0 21 LYS ( 24-) A 0 24 ARG ( 27-) A 0 26 SER ( 29-) A 0 47 SER ( 50-) A 0 49 ASP ( 52-) A 0 51 ALA ( 54-) A 0 54 LEU ( 57-) A 0 59 ASN ( 62-) A 0 61 HIS ( 64-) A 0 69 ASP ( 72-) A 0 70 SER ( 73-) A 0 72 ASP ( 75-) A 0 73 LYS ( 76-) A 0 77 LYS ( 80-) A 0 80 PRO ( 83-) A 0 82 ASP ( 85-) A 0 89 GLN ( 92-) A 0 100 GLN ( 103-) A 0 104 HIS ( 107-) A 0 108 LYS ( 111-) A 0 112 ALA ( 115-) A 0And so on for a total of 113 lines.
Standard deviation of omega values : 1.830
Warning: Unusual PRO puckering amplitudes
The proline residues listed in the table below have a puckering amplitude
that is outside of normal ranges. Puckering parameters were calculated by
the method of Cremer and Pople [REF]. Normal PRO rings have a puckering
amplitude Q between 0.20 and 0.45 Angstrom. If Q is lower than 0.20 Angstrom
for a PRO residue, this could indicate disorder between the two different
normal ring forms (with C-gamma below and above the ring, respectively). If
Q is higher than 0.45 Angstrom something could have gone wrong during the
refinement. 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]
151 PRO ( 155-) A 0.47 HIGH 197 PRO ( 201-) A 0.47 HIGH
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.
5 GLY ( 8-) A O <-> 9 GLY ( 12-) A N 0.24 2.46 INTRA 164 LYS ( 168-) A NZ <-> 258 HOH ( 323 ) A O 0.24 2.46 INTRA BF 126 ASP ( 130-) A OD2 <-> 129 LYS ( 133-) A NZ 0.19 2.51 INTRA 12 HIS ( 15-) A ND1 <-> 15 LYS ( 18-) A NZ 0.19 2.81 INTRA 157 VAL ( 161-) A CG1 <-> 221 LYS ( 225-) A CD 0.16 3.04 INTRA 45 SER ( 48-) A N <-> 77 LYS ( 80-) A O 0.13 2.57 INTRA 104 HIS ( 107-) A ND1 <-> 114 GLN ( 117-) A NE2 0.13 2.87 INTRA BL 121 ASN ( 124-) A N <-> 136 GLY ( 140-) A O 0.12 2.58 INTRA 93 HIS ( 96-) A NE2 <-> 258 HOH ( 327 ) A O 0.11 2.59 INTRA BL 24 ARG ( 27-) A NH2 <-> 203 VAL ( 207-) A O 0.10 2.60 INTRA BL 154 GLN ( 158-) A NE2 <-> 157 VAL ( 161-) A CG1 0.08 3.02 INTRA BL 54 LEU ( 57-) A O <-> 172 PHE ( 176-) A N 0.06 2.64 INTRA 71 GLN ( 74-) A O <-> 73 LYS ( 76-) A N 0.06 2.64 INTRA 196 THR ( 200-) A O <-> 199 LEU ( 203-) A N 0.06 2.64 INTRA 114 GLN ( 117-) A NE2 <-> 116 HIS ( 119-) A NE2 0.06 2.94 INTRA BL 203 VAL ( 207-) A CG1 <-> 204 THR ( 208-) A N 0.06 2.94 INTRA BL 150 LYS ( 154-) A O <-> 218 GLN ( 222-) A NE2 0.05 2.65 INTRA BL 94 TRP ( 97-) A NE1 <-> 237 MET ( 241-) A O 0.05 2.65 INTRA BL 26 SER ( 29-) A O <-> 242 ARG ( 246-) A NH1 0.05 2.65 INTRA BL 8 ASN ( 11-) A N <-> 9 GLY ( 12-) A N 0.04 2.56 INTRA B3 216 SER ( 220-) A O <-> 220 LEU ( 224-) A N 0.04 2.66 INTRA 29 ASP ( 32-) A OD1 <-> 108 LYS ( 111-) A N 0.04 2.66 INTRA 228 ASN ( 232-) A OD1 <-> 235 GLU ( 239-) A N 0.03 2.67 INTRA 123 LYS ( 127-) A N <-> 124 TYR ( 128-) A N 0.03 2.57 INTRA BL 91 HIS ( 94-) A NE2 <-> 258 HOH ( 327 ) A O 0.03 2.67 INTRA BL 69 ASP ( 72-) A OD2 <-> 120 TRP ( 123-) A NE1 0.02 2.68 INTRA BL 58 ASN ( 61-) A OD1 <-> 60 GLY ( 63-) A N 0.02 2.68 INTRA BL 2 TRP ( 5-) A CE3 <-> 13 TRP ( 16-) A CZ2 0.02 3.18 INTRA BL 36 LYS ( 39-) A CB <-> 251 GLN ( 255-) A NE2 0.02 3.08 INTRA 27 PRO ( 30-) A O <-> 245 GLN ( 249-) A N 0.02 2.68 INTRA BL 17 PHE ( 20-) A CA <-> 18 PRO ( 21-) A CD 0.02 2.78 INTRA BL 54 LEU ( 57-) A N <-> 66 GLU ( 69-) A O 0.01 2.69 INTRA 70 SER ( 73-) A N <-> 71 GLN ( 74-) A N 0.01 2.59 INTRA B3 89 GLN ( 92-) A O <-> 118 VAL ( 121-) A N 0.01 2.69 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.
7 HIS ( 10-) A -5.98 97 LEU ( 100-) A -5.39
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: Water molecules without hydrogen bonds
The water molecules listed in the table below do not form any hydrogen bonds,
neither with the protein or DNA/RNA, nor with other water molecules. This is
a strong indication of a refinement problem. The last number on each line is
the identifier of the water molecule in the input file.
258 HOH ( 333 ) A O
100 GLN ( 103-) A 251 GLN ( 255-) 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.
28 VAL ( 31-) A N 41 LEU ( 44-) A N 50 GLN ( 53-) A N 71 GLN ( 74-) A N 79 GLY ( 82-) A N 97 LEU ( 100-) A N 101 GLY ( 104-) A N 114 GLN ( 117-) A NE2 160 LEU ( 164-) A N 165 THR ( 169-) A OG1 178 ARG ( 182-) A N 178 ARG ( 182-) A NE 190 TYR ( 194-) A OH 196 THR ( 200-) A N 200 LEU ( 204-) A N 223 ARG ( 227-) A NH2 226 ASN ( 230-) A ND2 228 ASN ( 232-) A N 229 GLY ( 233-) A N 256 PHE ( 260-) 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.
116 HIS ( 119-) A NE2
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 : -0.434 2nd generation packing quality : -0.020 Ramachandran plot appearance : -2.702 chi-1/chi-2 rotamer normality : -2.753 Backbone conformation : -0.997
Bond lengths : 0.577 (tight) Bond angles : 0.849 Omega angle restraints : 0.333 (tight) Side chain planarity : 0.626 (tight) Improper dihedral distribution : 1.140 B-factor distribution : 1.505 (loose) Inside/Outside distribution : 0.950
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 : 2.50
Structure Z-scores, positive is better than average:
1st generation packing quality : 0.5 2nd generation packing quality : 0.8 Ramachandran plot appearance : -0.6 chi-1/chi-2 rotamer normality : -0.9 Backbone conformation : -0.6
Bond lengths : 0.577 (tight) Bond angles : 0.849 Omega angle restraints : 0.333 (tight) Side chain planarity : 0.626 (tight) Improper dihedral distribution : 1.140 B-factor distribution : 1.505 (loose) Inside/Outside distribution : 0.950 ==============
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.