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.
49 SER ( 73-) A - CB 49 SER ( 73-) A - OG 50 CYS ( 74-) A - CB 50 CYS ( 74-) A - SG 51 GLU ( 75-) A - CB 51 GLU ( 75-) A - CG 51 GLU ( 75-) A - CD 51 GLU ( 75-) A - OE1 51 GLU ( 75-) A - OE2 52 ASP ( 76-) A - CB 52 ASP ( 76-) A - CG 52 ASP ( 76-) A - OD1 52 ASP ( 76-) A - OD2
Plausible backbone 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. However, if a backbone atom is present in the PDB file, and its position seems 'logical' (i.e. normal bond lengths with all atoms it should be bound to, and those atoms exist normally) WHAT IF will set the occupancy to 1.0 if it believes that the full presence of this atom will be beneficial to the rest of the validation process. If you get weird errors at, or near, these atoms, please check by hand what is going on, and repair things intelligently before running this validation again.
49 SER ( 73-) A - CA 49 SER ( 73-) A - C 49 SER ( 73-) A - O 50 CYS ( 74-) A - N 50 CYS ( 74-) A - CA 50 CYS ( 74-) A - C 50 CYS ( 74-) A - O 51 GLU ( 75-) A - N 51 GLU ( 75-) A - CA 51 GLU ( 75-) A - C 51 GLU ( 75-) A - O 52 ASP ( 76-) A - N 52 ASP ( 76-) A - CA 52 ASP ( 76-) A - C 52 ASP ( 76-) A - O Residue with missing backbone atom(s) 237 ARG ( 261-) A -
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.
237 ARG ( 261-) 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: Missing atoms
The atoms listed in the table below are missing from the entry. If many atoms
are missing, the other checks can become less sensitive. Be aware that it
often happens that groups at the termini of DNA or RNA are really missing,
so that the absence of these atoms normally is neither an error nor the
result of poor electron density. Some of the atoms listed here might also be
listed by other checks, most noticeably by the options in the previous
section that list missing atoms in several categories. The plausible atoms
with zero occupancy are not listed here, as they already got assigned a
non-zero occupancy, and thus are no longer 'missing'.
237 ARG ( 261-) A O 237 ARG ( 261-) A CG 237 ARG ( 261-) A CD 237 ARG ( 261-) A NE 237 ARG ( 261-) A CZ 237 ARG ( 261-) A NH1 237 ARG ( 261-) A NH2
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.
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
Warning: Unusual bond lengths
The bond lengths listed in the table below were found to deviate more than 4
sigma from standard bond lengths (both standard values and sigmas for amino
acid residues have been taken from Engh and Huber [REF], for DNA they were
taken from Parkinson et al [REF]). In the table below for each unusual bond
the bond length and the number of standard deviations it differs from the
normal value is given.
Atom names starting with "-" belong to the previous residue in the chain. If the second atom name is "-SG*", the disulphide bridge has a deviating length.
70 HIS ( 94-) A ND1 CE1 1.38 4.6 72 HIS ( 96-) A CG CD2 1.41 4.6 72 HIS ( 96-) A ND1 CE1 1.39 5.2 95 HIS ( 119-) A ND1 CE1 1.38 4.6
70 HIS ( 94-) A CD2 CG ND1 114.60 8.5 72 HIS ( 96-) A CD2 CG ND1 110.35 4.3 95 HIS ( 119-) A CD2 CG ND1 111.97 5.9 184 THR ( 208-) A N CA C 99.75 -4.1
183 VAL ( 207-) A 5.92 12 LYS ( 36-) A 4.33 131 GLN ( 155-) A 4.17 184 THR ( 208-) A 4.09 143 VAL ( 167-) A 4.05
Tau angle RMS Z-score : 1.509
Error: Connections to aromatic rings out of plane
The atoms listed in the table below are connected to a planar aromatic group
in the sidechain of a protein residue but were found to deviate from the
least squares plane.
For all atoms that are connected to an aromatic side chain in a protein residue the distance of the atom to the least squares plane through the aromatic system was determined. This value was divided by the standard deviation from a distribution of similar values from a database of small molecule structures.
72 HIS ( 96-) A CB 4.64 Since there is no DNA and no protein with hydrogens, no uncalibrated planarity check was performed. Ramachandran Z-score : -2.562
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.
228 ARG ( 252-) A -2.8 196 PRO ( 220-) A -2.2 68 GLN ( 92-) A -2.2 62 ASN ( 86-) A -2.2 206 LEU ( 230-) A -2.1 190 THR ( 214-) A -2.1 153 GLY ( 177-) A -2.1 6 PRO ( 30-) A -2.1 154 ASP ( 178-) A -2.1 85 VAL ( 109-) 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 SER ( 29-) A PRO omega poor 58 GLY ( 82-) A Poor phi/psi 86 ASP ( 110-) A Poor phi/psi 147 ASP ( 171-) A Poor phi/psi 152 MET ( 176-) A Poor phi/psi 154 ASP ( 178-) A Poor phi/psi 177 PRO ( 201-) A PRO omega poor 179 LEU ( 203-) A Poor phi/psi 228 ARG ( 252-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -2.636
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!
3 ARG ( 27-) A 0 4 GLN ( 28-) A 0 5 SER ( 29-) A 0 11 TRP ( 35-) A 0 14 SER ( 38-) A 0 32 CYS ( 56-) A 0 33 ARG ( 57-) A 0 34 TYR ( 58-) A 0 36 TRP ( 60-) A 0 37 ASN ( 61-) A 0 38 THR ( 62-) A 0 40 HIS ( 64-) A 0 48 ASP ( 72-) A 0 49 SER ( 73-) A 0 50 CYS ( 74-) A 0 51 GLU ( 75-) A 0 53 SER ( 77-) A 0 55 ILE ( 79-) A 0 59 PRO ( 83-) A 0 67 LYS ( 91-) A 0 68 GLN ( 92-) A 0 75 ALA ( 99-) A 0 76 THR ( 100-) A 0 77 ASP ( 101-) A 0 78 GLU ( 102-) A 0And so on for a total of 112 lines.
Standard deviation of omega values : 1.420
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!
211 GLY ( 235-) A 1.53 80
18 PRO ( 42-) A 0.45 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.
159 CYS ( 183-) A SG <-> 239 HOH ( 295 ) A O 0.56 2.44 INTRA BL 164 CYS ( 188-) A SG <-> 239 HOH ( 297 ) A O 0.47 2.53 INTRA 9 ILE ( 33-) A O <-> 86 ASP ( 110-) A N 0.28 2.42 INTRA 9 ILE ( 33-) A N <-> 84 ALA ( 108-) A O 0.21 2.49 INTRA 3 ARG ( 27-) A CA <-> 230 ARG ( 254-) A NH1 0.17 2.93 INTRA 3 ARG ( 27-) A C <-> 230 ARG ( 254-) A NH1 0.17 2.93 INTRA 140 LEU ( 164-) A N <-> 141 PRO ( 165-) A CD 0.13 2.87 INTRA 37 ASN ( 61-) A O <-> 147 ASP ( 171-) A N 0.07 2.63 INTRA 6 PRO ( 30-) A O <-> 225 GLN ( 249-) A N 0.06 2.64 INTRA BL 153 GLY ( 177-) A C <-> 154 ASP ( 178-) A CG 0.06 3.04 INTRA BF 113 GLY ( 137-) A O <-> 182 SER ( 206-) A CB 0.06 2.74 INTRA 88 HIS ( 112-) A CE1 <-> 90 TYR ( 114-) A CE1 0.05 3.15 INTRA 235 SER ( 259-) A N <-> 236 PHE ( 260-) A N 0.04 2.56 INTRA BL 183 VAL ( 207-) A CG1 <-> 185 TRP ( 209-) A NE1 0.03 3.07 INTRA BL 8 ASN ( 32-) A OD1 <-> 87 GLY ( 111-) A N 0.03 2.67 INTRA 5 SER ( 29-) A O <-> 222 ARG ( 246-) A NH1 0.03 2.67 INTRA 52 ASP ( 76-) A O <-> 63 HIS ( 87-) A NE2 0.02 2.68 INTRA BL 104 TYR ( 128-) A OH <-> 114 GLU ( 138-) A N 0.02 2.68 INTRA BF 227 LEU ( 251-) A N <-> 239 HOH ( 302 ) A O 0.02 2.68 INTRA 76 THR ( 100-) A CG2 <-> 77 ASP ( 101-) A N 0.02 2.98 INTRA 27 TYR ( 51-) A OH <-> 98 HIS ( 122-) A NE2 0.01 2.69 INTRA BL 40 HIS ( 64-) A ND1 <-> 239 HOH ( 316 ) A O 0.01 2.69 INTRA 196 PRO ( 220-) A O <-> 200 SER ( 224-) A N 0.01 2.69 INTRA BL 199 LEU ( 223-) A CD1 <-> 203 ARG ( 227-) A NH2 0.01 3.09 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.
210 ARG ( 234-) A -6.39 114 GLU ( 138-) A -5.65 105 GLU ( 129-) A -5.24 50 CYS ( 74-) A -5.05
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.
112 VAL ( 136-) A -2.54
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.
239 HOH ( 295 ) A O Metal-coordinating Histidine residue 70 fixed to 1 Metal-coordinating Histidine residue 72 fixed to 1 Metal-coordinating Histidine residue 95 fixed to 1
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.
7 ILE ( 31-) A N 36 TRP ( 60-) A NE1 42 PHE ( 66-) A N 49 SER ( 73-) A N 62 ASN ( 86-) A ND2 71 PHE ( 95-) A N 76 THR ( 100-) A N 78 GLU ( 102-) A N 92 ALA ( 116-) A N 106 ASN ( 130-) A N 134 GLN ( 158-) A NE2 167 TYR ( 191-) A OH 176 THR ( 200-) A N 180 ALA ( 204-) A N 188 GLN ( 212-) A NE2 198 GLN ( 222-) A NE2 209 GLY ( 233-) A N 230 ARG ( 254-) A NH1 232 LEU ( 256-) A N Only metal coordination for 72 HIS ( 96-) 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.
188 GLN ( 212-) A OE1
51 GLU ( 75-) A H-bonding suggests Gln 86 ASP ( 110-) A H-bonding suggests Asn 154 ASP ( 178-) A H-bonding suggests Asn; but Alt-Rotamer 181 GLU ( 205-) 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 : -0.539 2nd generation packing quality : -1.545 Ramachandran plot appearance : -2.562 chi-1/chi-2 rotamer normality : -2.636 Backbone conformation : -1.087
Bond lengths : 0.515 (tight) Bond angles : 0.782 Omega angle restraints : 0.258 (tight) Side chain planarity : 0.455 (tight) Improper dihedral distribution : 0.900 B-factor distribution : 1.460 Inside/Outside distribution : 1.026
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 : 2.80
Structure Z-scores, positive is better than average:
1st generation packing quality : 0.7 2nd generation packing quality : 0.0 Ramachandran plot appearance : -0.1 chi-1/chi-2 rotamer normality : -0.5 Backbone conformation : -0.4
Bond lengths : 0.515 (tight) Bond angles : 0.782 Omega angle restraints : 0.258 (tight) Side chain planarity : 0.455 (tight) Improper dihedral distribution : 0.900 B-factor distribution : 1.460 Inside/Outside distribution : 1.026 ==============
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.