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 X-ray the coordinates must be located in density. Mobility or disorder sometimes cause this density to be so poor that the positions of the atoms cannot be determined. Crystallographers tend to leave out the atoms in such cases. This is not an error, albeit that we would prefer them to give it their best shot and provide coordinates with an occupancy of zero in cases where only a few atoms are involved. Anyway, several checks depend on the presence of the backbone atoms, so if you find errors in, or directly adjacent to, residues with missing backbone atoms, then please check by hand what is going on.
269 HIS ( 269-) A -
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: C-terminal nitrogen atoms detected.
It is becoming habit to indicate that a residue is not the true C-terminus
by including only the backbone N of the next residue. This has been
observed in this PDB file.
In X-ray the coordinates must be located in density. Mobility or disorder sometimes cause this density to be so poor that the positions of the atoms cannot be determined. Crystallographers tend to leave out the atoms in such cases. In many cases the N- or C-terminal residues are too disordered to see. In case of the N-terminus, you can see from the residue numbers if there are missing residues, but at the C-terminus this is impossible. Therefore, often the position of the backbone nitrogen of the first residue missing at the C-terminal end is calculated and added to indicate that there are missing residues. As a single N causes validation trouble, we remove these single-N-residues before doing the validation. But, if you get weird errors at, or near, the left-over incomplete C-terminal residue, please check by hand if a missing Oxt or removed N is the cause.
269 HIS ( 269-) A
Obviously, the temperature at which the X-ray data was collected has some importance too:
Crystal temperature (K) :288.000
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 : 1.915 over 3077 bonds
Average difference in B over a bond : 3.73
RMS difference in B over a bond : 4.71
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
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
| 0.999104 -0.000014 -0.000349| | -0.000014 0.997263 -0.000344| | -0.000349 -0.000344 0.996957|Proposed new scale matrix
| 0.017622 0.000000 0.000006| | 0.000000 0.012487 0.000004| | 0.000003 0.000003 0.008351|With corresponding cell
A = 56.748 B = 80.082 C = 119.740 Alpha= 90.040 Beta= 90.040 Gamma= 90.001
The CRYST1 cell dimensions
A = 56.800 B = 80.300 C = 120.100 Alpha= 90.000 Beta= 90.000 Gamma= 90.000
(Under-)estimated Z-score: 6.472
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.
7 HIS ( 7-) A CG ND1 CE1 109.76 4.2 89 HIS ( 89-) A CG ND1 CE1 109.61 4.0 117 HIS ( 117-) A CG ND1 CE1 109.77 4.2 297 TYR ( 298-) A N CA C 99.69 -4.1 402 ASN ( 403-) A N CA C 98.31 -4.6 441 HIS ( 442-) A CG ND1 CE1 109.71 4.1 447 ASN ( 448-) A -C N CA 129.54 4.4
22 GLN ( 22-) A 4.75 256 ALA ( 256-) A 4.65 139 LEU ( 139-) A 4.53 129 ASN ( 129-) A 4.52 297 TYR ( 298-) A 4.39 402 ASN ( 403-) A 4.39 135 GLN ( 135-) A 4.24
Tau angle RMS Z-score : 1.603
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.
328 PHE ( 329-) A -3.0 364 THR ( 365-) A -2.7 140 VAL ( 140-) A -2.5 105 ASN ( 105-) A -2.3 37 PRO ( 37-) A -2.1 280 GLY ( 281-) A -2.1 303 SER ( 304-) A -2.1 302 SER ( 303-) 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 THR ( 3-) A Poor phi/psi 10 GLU ( 10-) A Poor phi/psi 57 LEU ( 57-) A Poor phi/psi 106 LYS ( 106-) A Poor phi/psi 307 HIS ( 308-) A Poor phi/psi 308 GLY ( 309-) A Poor phi/psi 322 ASN ( 323-) A Poor phi/psi 328 PHE ( 329-) A Poor phi/psi 329 ALA ( 330-) A Poor phi/psi 331 ASN ( 332-) A Poor phi/psi 334 CYS ( 335-) A Poor phi/psi 364 THR ( 365-) A Poor phi/psi 365 ASN ( 366-) A Poor phi/psi 412 ALA ( 413-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -2.532
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!
9 PHE ( 9-) A 0 10 GLU ( 10-) A 0 11 TRP ( 11-) A 0 22 GLN ( 22-) A 0 23 TYR ( 23-) A 0 24 LEU ( 24-) A 0 30 ALA ( 30-) A 0 36 PRO ( 36-) A 0 37 PRO ( 37-) A 0 38 ASN ( 38-) A 0 40 HIS ( 40-) A 0 41 ILE ( 41-) A 0 42 THR ( 42-) A 0 47 TRP ( 47-) A 0 50 TYR ( 50-) A 0 51 GLN ( 51-) A 0 52 PRO ( 52-) A 0 55 TYR ( 55-) A 0 57 LEU ( 57-) A 0 58 GLN ( 58-) A 0 59 SER ( 59-) A 0 63 ASN ( 63-) A 0 89 HIS ( 89-) A 0 90 MET ( 90-) A 0 92 ALA ( 92-) A 0And so on for a total of 203 lines.
Standard deviation of omega values : 1.742
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]
36 PRO ( 36-) A 0.46 HIGH 113 PRO ( 113-) A 0.46 HIGH
109 PRO ( 109-) A -117.9 half-chair C-delta/C-gamma (-126 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.
177 LYS ( 177-) A NZ <-> 450 HOH (1267 ) A O 0.22 2.48 INTRA 178 HIS ( 178-) A NE2 <-> 450 HOH (1222 ) A O 0.14 2.56 INTRA BL 412 ALA ( 413-) A O <-> 447 ASN ( 448-) A N 0.12 2.58 INTRA BF 224 LYS ( 224-) A NZ <-> 450 HOH (1077 ) A O 0.07 2.63 INTRA 106 LYS ( 106-) A NZ <-> 117 HIS ( 117-) A O 0.05 2.65 INTRA 262 ASN ( 262-) A OD1 <-> 265 ASN ( 265-) A N 0.05 2.65 INTRA BL 353 ASP ( 354-) A OD2 <-> 442 LYS ( 443-) A NZ 0.05 2.65 INTRA 299 LYS ( 300-) A NZ <-> 449 CL ( 900-) A CL 0.05 3.05 INTRA 172 ARG ( 172-) A NH1 <-> 174 ASP ( 174-) A OD1 0.03 2.67 INTRA BL 14 GLN ( 14-) A NE2 <-> 450 HOH (1281 ) A O 0.03 2.67 INTRA 248 TRP ( 248-) A N <-> 249 GLY ( 249-) A N 0.02 2.58 INTRA BL 127 TYR ( 127-) A O <-> 208 ALA ( 208-) A N 0.02 2.68 INTRA
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.
204 GLN ( 204-) A -6.48 60 ARG ( 60-) A -6.04 316 ASN ( 317-) A -5.73 26 PRO ( 26-) A -5.49 267 ARG ( 267-) A -5.46 248 TRP ( 248-) A -5.44 235 ASN ( 235-) A -5.43 243 ASN ( 243-) A -5.34 22 GLN ( 22-) A -5.31 234 ARG ( 234-) A -5.30 136 ASN ( 136-) A -5.04
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.
121 THR ( 121-) A -3.24 306 PHE ( 307-) A -2.65
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.
450 HOH (1285 ) A O Marked this atom as acceptor 449 CL ( 900-) A CL Strange metal coordination for HIS 178
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.
3 THR ( 3-) A N 4 THR ( 4-) A N 31 ALA ( 31-) A N 38 ASN ( 38-) A N 38 ASN ( 38-) A ND2 47 TRP ( 47-) A N 89 HIS ( 89-) A N 105 ASN ( 105-) A N 110 ILE ( 110-) A N 127 TYR ( 127-) A N 204 GLN ( 204-) A N 237 SER ( 237-) A N 245 GLY ( 245-) A N 264 ASP ( 264-) A N 268 GLY ( 268-) A N 287 ASN ( 288-) A ND2 376 GLY ( 377-) A N 409 SER ( 410-) A OG 441 HIS ( 442-) A N Only metal coordination for 88 ASN ( 88-) A OD1
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.
7 HIS ( 7-) A NE2
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+.
448 CA ( 800-) A 0.75 0.99 Scores about as good as NA
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 : -0.721 2nd generation packing quality : -1.421 Ramachandran plot appearance : -2.036 chi-1/chi-2 rotamer normality : -2.532 Backbone conformation : -1.479
Bond lengths : 0.500 (tight) Bond angles : 0.749 Omega angle restraints : 0.317 (tight) Side chain planarity : 0.526 (tight) Improper dihedral distribution : 0.986 B-factor distribution : 1.915 (loose) Inside/Outside distribution : 0.976
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 : 2.40
Structure Z-scores, positive is better than average:
1st generation packing quality : 0.1 2nd generation packing quality : -0.3 Ramachandran plot appearance : -0.2 chi-1/chi-2 rotamer normality : -0.8 Backbone conformation : -1.2
Bond lengths : 0.500 (tight) Bond angles : 0.749 Omega angle restraints : 0.317 (tight) Side chain planarity : 0.526 (tight) Improper dihedral distribution : 0.986 B-factor distribution : 1.915 (loose) Inside/Outside distribution : 0.976 ==============
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.