WHAT IF Check report

This file was created 2011-12-17 from WHAT_CHECK output by a conversion script. If you are new to WHAT_CHECK, please study the pdbreport pages. There also exists a legend to the output.

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.

Verification log for pdb1nmb.ent

Checks that need to be done early-on in validation

Warning: Matthews Coefficient (Vm) high

The Matthews coefficient [REF] is defined as the density of the protein structure in cubic Angstroms per Dalton. Normal values are between 1.5 (tightly packed, little room for solvent) and 4.0 (loosely packed, much space for solvent). Some very loosely packed structures can get values a bit higher than that.

Very high numbers are most often caused by giving the wrong value for Z on the CRYST1 card (or not giving this number at all), but can also result from large fractions missing out of the molecular weight (e.g. a lot of UNK residues, or DNA/RNA missing from virus structures).

Molecular weight of all polymer chains: 69942.992
Volume of the Unit Cell V= 4503088.5
Space group multiplicity: 16
No NCS symmetry matrices (MTRIX records) found in PDB file
Matthews coefficient for observed atoms and Z a bit high: Vm= 4.024
Vm by authors and this calculated Vm agree only marginally
Matthews coefficient read from REMARK 280 Vm= 3.610

Warning: Ligands for which a topology was generated automatically

The topology for the ligands in the table below were determined automatically. WHAT IF uses a local copy of Daan van Aalten's Dundee PRODRG server to automatically generate topology information for ligands. For this PDB file that seems to have gone fine, but be aware that automatic topology generation is a complicated task. So, if you get messages that you fail to understand or that you believe are wrong, and one of these ligands is involved, then check the ligand topology first.

 627 MAN   ( 472-)  N  -
 628 MAN   ( 473-)  N  -
 629 MAN   ( 474-)  N  -
 631 MAN   ( 475-)  N  -
 632 BMA   ( 471-)  N  -

Administrative problems that can generate validation failures

Warning: Groups attached to potentially hydrogenbonding atoms

Residues were observed with groups attached to (or very near to) atoms that potentially can form hydrogen bonds. WHAT IF is not very good at dealing with such exceptional cases (Mainly because it's author is not...). So be warned that the hydrogenbonding-related analyses of these residues might be in error.

For example, an aspartic acid can be protonated on one of its delta oxygens. This is possible because the one delta oxygen 'helps' the other one holding that proton. However, if a delta oxygen has a group bound to it, then it can no longer 'help' the other delta oxygen bind the proton. However, both delta oxygens, in principle, can still be hydrogen bond acceptors. Such problems can occur in the amino acids Asp, Glu, and His. I have opted, for now to simply allow no hydrogen bonds at all for any atom in any side chain that somewhere has a 'funny' group attached to it. I know this is wrong, but there are only 12 hours in a day.

 620 NAG   ( 469A)  N  -   O4  bound to  621 NAG   ( 470B)  N  -   C1
 621 NAG   ( 470B)  N  -   O4  bound to  632 BMA   ( 471-)  N  -   C1

Warning: Plausible side chain atoms detected with zero occupancy

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.

 395 THR   (   7-)  L  -   CB
 395 THR   (   7-)  L  -   OG1
 395 THR   (   7-)  L  -   CG2
 415 GLN   (  27-)  L  -   CB
 415 GLN   (  27-)  L  -   CG
 415 GLN   (  27-)  L  -   CD
 415 GLN   (  27-)  L  -   OE1
 415 GLN   (  27-)  L  -   NE2
 433 LYS   (  45-)  L  -   CB
 433 LYS   (  45-)  L  -   CG
 433 LYS   (  45-)  L  -   CD
 433 LYS   (  45-)  L  -   CE
 433 LYS   (  45-)  L  -   NZ
 469 GLU   (  81-)  L  -   CB
 469 GLU   (  81-)  L  -   CG
 469 GLU   (  81-)  L  -   CD
 469 GLU   (  81-)  L  -   OE1
 469 GLU   (  81-)  L  -   OE2
 495 ARG   ( 107-)  L  -   CB
 495 ARG   ( 107-)  L  -   CG
 495 ARG   ( 107-)  L  -   CD
 495 ARG   ( 107-)  L  -   NE
 495 ARG   ( 107-)  L  -   CZ
 495 ARG   ( 107-)  L  -   NH1
 495 ARG   ( 107-)  L  -   NH2
 497 ALA   ( 109-)  L  -   CB
 508 LEU   (  11-)  H  -   CB
 508 LEU   (  11-)  H  -   CG
 508 LEU   (  11-)  H  -   CD1
 508 LEU   (  11-)  H  -   CD2
 538 PRO   (  41-)  H  -   CB
 538 PRO   (  41-)  H  -   CG
 538 PRO   (  41-)  H  -   CD
 585 SER   (  84-)  H  -   CB
 585 SER   (  84-)  H  -   OG
 601 ARG   ( 100-)  H  -   CB
 601 ARG   ( 100-)  H  -   CG
 601 ARG   ( 100-)  H  -   CD
 601 ARG   ( 100-)  H  -   NE
 601 ARG   ( 100-)  H  -   CZ
 601 ARG   ( 100-)  H  -   NH1
 601 ARG   ( 100-)  H  -   NH2
 618 SER   ( 112-)  H  -   CB
 618 SER   ( 112-)  H  -   OG
 619 SER   ( 113-)  H  -   CB
 619 SER   ( 113-)  H  -   OG

Warning: Plausible backbone atoms detected with zero occupancy

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.

 395 THR   (   7-)  L  -   N
 395 THR   (   7-)  L  -   CA
 395 THR   (   7-)  L  -   C
 395 THR   (   7-)  L  -   O
 497 ALA   ( 109-)  L  -   N
 497 ALA   ( 109-)  L  -   CA
 497 ALA   ( 109-)  L  -   C
 497 ALA   ( 109-)  L  -   O
 538 PRO   (  41-)  H  -   N
 538 PRO   (  41-)  H  -   CA
 538 PRO   (  41-)  H  -   C
 538 PRO   (  41-)  H  -   O
 618 SER   ( 112-)  H  -   N
 618 SER   ( 112-)  H  -   CA
 618 SER   ( 112-)  H  -   C
 618 SER   ( 112-)  H  -   O
 619 SER   ( 113-)  H  -   N
 619 SER   ( 113-)  H  -   CA
 619 SER   ( 113-)  H  -   C
 619 SER   ( 113-)  H  -   O
 625 ALA   ( 109-)  L  -   O''
 626 SER   ( 113-)  H  -   O''

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: N

Note: Ramachandran plot

Chain identifier: L

Note: Ramachandran plot

Chain identifier: H

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:

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 5 percent of buried atoms has low B-factor

For normal protein structures, no more than about 1 percent of the B factors of buried atoms is below 5.0. The fact that this value is much higher in the current structure could be a signal that the B-factors were restraints or constraints to too-low values, misuse of B-factor field in the PDB file, or a TLS/scaling problem. If the average B factor is low too, it is probably a low temperature structure determination.

Percentage of buried atoms with B less than 5 : 45.82

Note: B-factor plot

The average atomic B-factor per residue is plotted as function of the residue number.

Chain identifier: N

Note: B-factor plot

Chain identifier: L

Note: B-factor plot

Chain identifier: H

Geometric checks

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

 |  0.998017 -0.000132 -0.000285|
 | -0.000132  0.998392  0.000316|
 | -0.000285  0.000316  1.001558|
Proposed new scale matrix

 |  0.005915  0.000000  0.000002|
 |  0.000000  0.005913 -0.000002|
 |  0.000002 -0.000002  0.006363|
With corresponding cell

    A    = 169.069  B   = 169.133  C    = 157.156
    Alpha=  89.964  Beta=  90.033  Gamma=  90.002

The CRYST1 cell dimensions

    A    = 169.400  B   = 169.400  C    = 156.900
    Alpha=  90.000  Beta=  90.000  Gamma=  90.000

Variance: 30.946
(Under-)estimated Z-score: 4.100

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.

  44 ASP   ( 125-)  N      N    CA   C    98.29   -4.6
  48 CYS   ( 129-)  N      N    CA   C    98.91   -4.4
 146 GLN   ( 226-)  N      N    CA   C   124.65    4.8
 219 ASN   ( 299-)  N      N    CA   C    97.33   -5.0
 224 GLN   ( 304-)  N      N    CA   C    99.50   -4.2
 232 HIS   ( 312-)  N      CG   ND1  CE1 109.62    4.0
 241 VAL   ( 321-)  N      N    CA   C    98.84   -4.4
 339 ARG   ( 419-)  N      CB   CG   CD  104.48   -4.8
 428 PRO   (  40-)  L      N    CA   C   122.03    4.1
 443 HIS   (  55-)  L      CG   ND1  CE1 109.62    4.0
 558 ASN   (  60-)  H      N    CA   C    98.47   -4.5
 593 CYS   (  92-)  H      N    CA   C    99.97   -4.0
 623 NAG   ( 477-)  N      N2   C2   C1  100.26   -4.3

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.

 146 GLN   ( 226-)  N    5.22
 528 ASN   (  31-)  H    4.87
 219 ASN   ( 299-)  N    4.71
  48 CYS   ( 129-)  N    4.50
 168 GLY   ( 248-)  N    4.42
 558 ASN   (  60-)  H    4.34
 241 VAL   ( 321-)  N    4.33
 216 GLN   ( 296-)  N    4.09
 593 CYS   (  92-)  H    4.05
 224 GLN   ( 304-)  N    4.01
  19 TYR   ( 100-)  N    4.01
  44 ASP   ( 125-)  N    4.01

Warning: High tau angle deviations

The RMS Z-score for the tau angles (N-Calpha-C) in the structure is too high. For well refined structures this number is expected to be near 1.0. The fact that it is higher than 1.5 worries us. However, we determined the tau normal distributions from 500 high-resolution X-ray structures, rather than from CSD data, so we cannot be 100 percent certain about these numbers.

Tau angle RMS Z-score : 1.648

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.

 343 TYR   ( 423-)  N      CB   6.01
 343 TYR   ( 423-)  N      OH   4.26
Since there is no DNA and no protein with hydrogens, no uncalibrated
planarity check was performed.
 Ramachandran Z-score : -3.435

Torsion-related checks

Warning: Ramachandran Z-score low

The score expressing how well the backbone conformations of all residues correspond to the known allowed areas in the Ramachandran plot is a bit low.

Ramachandran Z-score : -3.435

Warning: Torsion angle evaluation shows unusual residues

The residues listed in the table below contain bad or abnormal torsion angles.

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.

 140 ARG   ( 220-)  N    -2.9
 496 ARG   ( 108-)  L    -2.7
 232 HIS   ( 312-)  N    -2.6
 395 THR   (   7-)  L    -2.6
  37 ARG   ( 118-)  N    -2.6
 384 ILE   ( 464-)  N    -2.5
 466 LEU   (  78-)  L    -2.5
 287 ILE   ( 368-)  N    -2.5
  57 THR   ( 138-)  N    -2.5
 418 SER   (  30-)  L    -2.5
 204 GLN   ( 284-)  N    -2.4
 447 PRO   (  59-)  L    -2.4
 145 THR   ( 225-)  N    -2.4
 438 TYR   (  50-)  L    -2.4
 431 THR   (  43-)  L    -2.4
 456 GLY   (  68-)  L    -2.4
 266 ASN   ( 347-)  N    -2.4
 184 LYS   ( 264-)  N    -2.4
 101 THR   ( 181-)  N    -2.4
 231 THR   ( 311-)  N    -2.4
 524 TYR   (  27-)  H    -2.4
 233 THR   ( 313-)  N    -2.4
 144 ARG   ( 224-)  N    -2.3
 515 VAL   (  18-)  H    -2.3
 582 SER   (  82-)  H    -2.3
And so on for a total of 58 lines.

Warning: Backbone evaluation reveals unusual conformations

The residues listed in the table below have abnormal backbone torsion angles.

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.

   6 LEU   (  87-)  N  Poor phi/psi
  23 ASN   ( 104-)  N  Poor phi/psi
  31 SER   ( 112-)  N  Poor phi/psi
  37 ARG   ( 118-)  N  Poor phi/psi
  40 TYR   ( 121-)  N  Poor phi/psi
  45 PRO   ( 126-)  N  Poor phi/psi
  83 SER   ( 164-)  N  Poor phi/psi
  95 CYS   ( 175-)  N  Poor phi/psi
 100 SER   ( 180-)  N  Poor phi/psi
 101 THR   ( 181-)  N  Poor phi/psi
 128 ASN   ( 208-)  N  Poor phi/psi
 129 ARG   ( 209-)  N  Poor phi/psi
 132 VAL   ( 212-)  N  Poor phi/psi
 145 THR   ( 225-)  N  Poor phi/psi
 147 GLU   ( 227-)  N  Poor phi/psi
 184 LYS   ( 264-)  N  Poor phi/psi
 203 GLU   ( 283-)  N  Poor phi/psi
 204 GLN   ( 284-)  N  Poor phi/psi
 211 CYS   ( 291-)  N  Poor phi/psi
 230 MET   ( 310-)  N  Poor phi/psi
 232 HIS   ( 312-)  N  Poor phi/psi
 235 GLN   ( 315-)  N  Poor phi/psi
 245 ASN   ( 325-)  N  PRO omega poor
 265 ASN   ( 346-)  N  Poor phi/psi
 266 ASN   ( 347-)  N  Poor phi/psi
 278 ASN   ( 359-)  N  Poor phi/psi
 306 ARG   ( 387-)  N  Poor phi/psi
 313 GLN   ( 395-)  N  Poor phi/psi
 322 SER   ( 404-)  N  Poor phi/psi
 328 PHE   ( 410-)  N  Poor phi/psi
 350 ARG   ( 430-)  N  PRO omega poor
 396 THR   (   8-)  L  Poor phi/psi
 419 ASN   (  31-)  L  Poor phi/psi
 420 TYR   (  32-)  L  Poor phi/psi
 430 GLY   (  42-)  L  Poor phi/psi
 438 TYR   (  50-)  L  Poor phi/psi
 439 THR   (  51-)  L  Poor phi/psi
 456 GLY   (  68-)  L  Poor phi/psi
 482 LEU   (  94-)  L  PRO omega poor
 562 LYS   (  64-)  H  Poor phi/psi
 574 ASN   (  76-)  H  Poor phi/psi
 582 SER   (  82-)  H  Poor phi/psi
 589 ALA   (  88-)  H  Poor phi/psi
 599 SER   (  98-)  H  Poor phi/psi
 chi-1/chi-2 correlation Z-score : -4.062

Error: chi-1/chi-2 angle correlation Z-score very low

The score expressing how well the chi-1/chi-2 angles of all residues correspond to the populated areas in the database is very low.

chi-1/chi-2 correlation Z-score : -4.062

Warning: Unusual backbone conformations

For the residues listed in the table below, the backbone formed by itself and two neighbouring residues on either side is in a conformation that is not seen very often in the database of solved protein structures. The number given in the table is the number of similar backbone conformations in the database with the same amino acid in the centre.

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!

   7 THR   (  88-)  N      0
  12 THR   (  93-)  N      0
  14 ASN   (  95-)  N      0
  19 TYR   ( 100-)  N      0
  30 ASP   ( 111-)  N      0
  31 SER   ( 112-)  N      0
  32 ASP   ( 113-)  N      0
  37 ARG   ( 118-)  N      0
  38 GLU   ( 119-)  N      0
  39 PRO   ( 120-)  N      0
  40 TYR   ( 121-)  N      0
  46 ASP   ( 127-)  N      0
  47 GLU   ( 128-)  N      0
  63 HIS   ( 144-)  N      0
  65 ASN   ( 146-)  N      0
  69 HIS   ( 150-)  N      0
  70 ASP   ( 151-)  N      0
  71 ARG   ( 152-)  N      0
  72 SER   ( 153-)  N      0
  74 TYR   ( 155-)  N      0
  80 TRP   ( 161-)  N      0
  82 LEU   ( 163-)  N      0
  83 SER   ( 164-)  N      0
  87 THR   ( 168-)  N      0
  94 GLU   ( 174-)  N      0
And so on for a total of 336 lines.

Warning: Omega angles too tightly restrained

The omega angles for trans-peptide bonds in a structure are expected to give a gaussian distribution with the average around +178 degrees and a standard deviation around 5.5 degrees. These expected values were obtained from very accurately determined structures. Many protein structures are too tightly restrained. This seems to be the case with the current structure too, as the observed standard deviation is below 4.0 degrees.

Standard deviation of omega values : 1.831

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]

  45 PRO   ( 126-)  N    0.45 HIGH
 246 PRO   ( 326-)  N    0.46 HIGH
 251 PRO   ( 331-)  N    0.46 HIGH
 259 PRO   ( 340-)  N    0.46 HIGH
 351 PRO   ( 431-)  N    0.47 HIGH
 428 PRO   (  40-)  L    0.45 HIGH

Warning: Unusual PRO puckering phases

The proline residues listed in the table below have a puckering phase that is not expected to occur in protein structures. Puckering parameters were calculated by the method of Cremer and Pople [REF]. Normal PRO rings approximately show a so-called envelope conformation with the C-gamma atom above the plane of the ring (phi=+72 degrees), or a half-chair conformation with C-gamma below and C-beta above the plane of the ring (phi=-90 degrees). If phi deviates strongly from these values, this is indicative of a very strange conformation for a PRO residue, and definitely requires a manual check of the data. 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].

 428 PRO   (  40-)  L  -121.6 half-chair C-delta/C-gamma (-126 degrees)

Bump checks

Error: Abnormally short interatomic distances

The pairs of atoms listed in the table below have an unusually short interactomic distance; each bump is listed in only one direction.

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.

 621 NAG   ( 470-)  N      O4  <->  632 BMA   ( 471-)  N      C1     1.01    1.39  INTRA B3
 621 NAG   ( 470-)  N      C4  <->  632 BMA   ( 471-)  N      C1     0.75    2.45  INTRA
 348 ARG   ( 428-)  N      NH2 <->  382 ALA   ( 462-)  N      O      0.28    2.42  INTRA BL
 449 ARG   (  61-)  L      NH2 <->  470 ASP   (  82-)  L      CG     0.27    2.83  INTRA
 512 GLY   (  15-)  H      N   <->  583 LEU   (  82-)  H      O      0.27    2.43  INTRA
 220 ARG   ( 300-)  N      NH2 <->  268 VAL   ( 349-)  N      O      0.22    2.48  INTRA BL
  49 ARG   ( 130-)  N      NH1 <->   80 TRP   ( 161-)  N      N      0.22    2.63  INTRA BL
  92 ARG   ( 172-)  N      CD  <->  129 ARG   ( 209-)  N      NH2    0.22    2.88  INTRA
 427 ASN   (  39-)  L      ND2 <->  431 THR   (  43-)  L      CB     0.21    2.89  INTRA
  43 CYS   ( 124-)  N      N   <->  633 HOH   ( 503 )  N      O      0.21    2.49  INTRA BL
  44 ASP   ( 125-)  N      OD1 <->  109 ARG   ( 189-)  N      NH2    0.21    2.49  INTRA BL
  16 TRP   (  97-)  N      CZ3 <->  280 TRP   ( 361-)  N      CD2    0.20    3.00  INTRA
   8 LYS   (  89-)  N      C   <->  204 GLN   ( 284-)  N      NE2    0.20    2.90  INTRA
 393 THR   (   5-)  L      O   <->  412 ARG   (  24-)  L      N      0.19    2.51  INTRA
  68 ILE   ( 149-)  N      CG2 <->   69 HIS   ( 150-)  N      N      0.19    2.81  INTRA BL
 103 CYS   ( 183-)  N      N   <->  150 CYS   ( 230-)  N      SG     0.17    3.13  INTRA BL
  53 LEU   ( 134-)  N      CB  <->   75 ARG   ( 156-)  N      NH2    0.16    2.94  INTRA BL
 419 ASN   (  31-)  L      ND2 <->  439 THR   (  51-)  L      OG1    0.16    2.54  INTRA BL
 545 ILE   (  48-)  H      O   <->  558 ASN   (  60-)  H      N      0.16    2.54  INTRA BL
  92 ARG   ( 172-)  N      NH1 <->  633 HOH   ( 479 )  N      O      0.16    2.54  INTRA BL
  80 TRP   ( 161-)  N      NE1 <->   84 SER   ( 165-)  N      O      0.15    2.55  INTRA BL
 276 GLY   ( 357-)  N      O   <->  279 THR   ( 360-)  N      CG2    0.15    2.65  INTRA BL
 393 THR   (   5-)  L      N   <->  412 ARG   (  24-)  L      O      0.15    2.55  INTRA BL
 103 CYS   ( 183-)  N      SG  <->  152 CYS   ( 232-)  N      SG     0.15    3.30  INTRA BL
   4 ASN   (  85-)  N      CG  <->    5 ASN   (  86-)  N      N      0.14    2.86  INTRA BL
And so on for a total of 137 lines.

Packing, accessibility and threading

Note: Inside/Outside RMS Z-score plot

The Inside/Outside distribution normality RMS Z-score over a 15 residue window is plotted as function of the residue number. High areas in the plot (above 1.5) indicate unusual inside/outside patterns.

Chain identifier: N

Note: Inside/Outside RMS Z-score plot

Chain identifier: L

Note: Inside/Outside RMS Z-score plot

Chain identifier: H

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.

 496 ARG   ( 108-)  L      -8.36
 611 GLN   ( 105-)  H      -6.46
 495 ARG   ( 107-)  L      -6.44
 334 GLU   ( 414-)  N      -6.17
 540 GLN   (  43-)  H      -6.09
  71 ARG   ( 152-)  N      -6.08
  89 TYR   ( 169-)  N      -5.82
 375 GLN   ( 455-)  N      -5.77
 107 ARG   ( 187-)  N      -5.61
 372 PHE   ( 452-)  N      -5.23
 445 GLU   (  57-)  L      -5.14
 600 TYR   (  99-)  H      -5.06

Warning: Abnormal packing environment for sequential residues

A stretch of at least three sequential residues with a questionable packing environment was found. This could indicate that these residues are part of a strange loop. It might also be an indication of misthreading in the density. However, it can also indicate that one or more residues in this stretch have other problems such as, for example, missing atoms, very weird angles or bond lengths, etc.

The table below lists the first and last residue in each stretch found, as well as the average residue score of the series.

 495 ARG   ( 107-)  L       497 - ALA    109- ( L)         -6.41

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 packing.

Chain identifier: N

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 packing.

Chain identifier: L

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 packing.

Chain identifier: H

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.

 313 GLN   ( 395-)  N   -2.64

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: N

Note: Second generation quality Z-score plot

Chain identifier: L

Note: Second generation quality Z-score plot

Chain identifier: H

Water, ion, and hydrogenbond related checks

Warning: Water molecules need moving

The water molecules listed in the table below were found to be significantly closer to a symmetry related non-water molecule than to the ones given in the coordinate file. For optimal viewing convenience revised coordinates for these water molecules should be given.

The number in brackets is the identifier of the water molecule in the input file. Suggested coordinates are also given in the table. Please note that alternative conformations for protein residues are not taken into account for this calculation. If you are using WHAT IF / WHAT-CHECK interactively, then the moved waters can be found in PDB format in the file: MOVEDH2O.pdb.

 633 HOH   ( 494 )  N      O     61.78   74.66   44.48

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.

 633 HOH   ( 543 )  N      O
 633 HOH   ( 544 )  N      O
Bound group on Asn; dont flip    5 ASN  (  86-) N
Bound to:  623 NAG  ( 477-) N
Bound group on Asn; dont flip   65 ASN  ( 146-) N
Bound to:  622 NAG  ( 476-) N

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.

  17 HIS   (  98-)  N
  23 ASN   ( 104-)  N
  63 HIS   ( 144-)  N
  73 GLN   ( 154-)  N
 136 ASN   ( 216-)  N
 257 ASN   ( 338-)  N
 266 ASN   ( 347-)  N
 313 GLN   ( 395-)  N
 377 ASN   ( 457-)  N
 611 GLN   ( 105-)  H

Warning: Buried unsatisfied hydrogen bond donors

The buried hydrogen bond donors listed in the table below have a hydrogen atom that is not involved in a hydrogen bond in the optimized hydrogen bond network.

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.

  16 TRP   (  97-)  N      NE1
  21 LYS   ( 102-)  N      N
  25 VAL   ( 106-)  N      N
  37 ARG   ( 118-)  N      NE
  40 TYR   ( 121-)  N      OH
  41 VAL   ( 122-)  N      N
  46 ASP   ( 127-)  N      N
  49 ARG   ( 130-)  N      NH1
  57 THR   ( 138-)  N      N
  63 HIS   ( 144-)  N      N
  72 SER   ( 153-)  N      N
  75 ARG   ( 156-)  N      N
  76 ASP   ( 157-)  N      N
  83 SER   ( 164-)  N      N
  99 SER   ( 179-)  N      N
 102 SER   ( 182-)  N      N
 116 GLY   ( 196-)  N      N
 119 ASN   ( 199-)  N      N
 121 ALA   ( 201-)  N      N
 130 ARG   ( 210-)  N      N
 140 ARG   ( 220-)  N      NH1
 144 ARG   ( 224-)  N      NH2
 148 SER   ( 228-)  N      N
 155 GLY   ( 235-)  N      N
 172 THR   ( 252-)  N      OG1
And so on for a total of 69 lines.

Warning: Buried unsatisfied hydrogen bond acceptors

The buried side-chain hydrogen bond acceptors listed in the table below are not involved in a hydrogen bond in the optimized hydrogen bond network.

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.

  76 ASP   ( 157-)  N      OD1
 196 GLU   ( 276-)  N      OE1
 232 HIS   ( 312-)  N      ND1
 249 ASN   ( 329-)  N      OD1
 470 ASP   (  82-)  L      OD2
 587 ASP   (  86-)  H      OD2

Warning: No crystallisation information

No, or very inadequate, crystallisation information was observed upon reading the PDB file header records. This information should be available in the form of a series of REMARK 280 lines. Without this information a few things, such as checking ions in the structure, cannot be performed optimally.

Warning: Unusual ion packing

We implemented the ion valence determination method of Brown and Wu [REF] similar to Nayal and Di Cera [REF]. See also Mueller, Koepke and Sheldrick [REF]. It must be stated that the validation of ions in PDB files is very difficult. Ideal ion-ligand distances often differ no more than 0.1 Angstrom, and in a 2.0 Angstrom resolution structure 0.1 Angstrom is not very much. Nayal and Di Cera showed that this method has great potential, but the method has not been validated. Part of our implementation (comparing ion types) is even fully new and despite that we see it work well in the few cases that are trivial, we must emphasize that this validation method is untested. See: swift.cmbi.ru.nl/teach/theory/ for a detailed explanation.

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+.

 630  CA   ( 478-)  N     0.67   0.89 Scores about as good as NA

Warning: Unusual water packing

We implemented the ion valence determination method of Brown and Wu [REF] similar to Nayal and Di Cera [REF] and Mueller, Koepke and Sheldrick [REF]. It must be stated that the validation of ions in PDB files is very difficult. Ideal ion-ligand distances often differ no more than 0.1 Angstrom, and in a 2.0 Angstrom resolution structure 0.1 Angstrom is not very much. Nayal and Di Cera showed that this method nevertheless has great potential for detecting water molecules that actually should be metal ions. The method has not been extensively validated, though. Part of our implementation (comparing waters with multiple ion types) is even fully new and despite that we see it work well in the few cases that are trivial, we must emphasize that this method is untested.

The score listed is the valency score. This number should be close to (preferably a bit above) 1.0 for the suggested ion to be a likely alternative for the water molecule. Ions listed in brackets are good alternate choices. *1 indicates that the suggested ion-type has been observed elsewhere in the PDB file too. *2 indicates that the suggested ion-type has been observed in the REMARK 280 cards of the PDB file. Ion-B and ION-B indicate that the B-factor of this water is high, or very high, respectively. H2O-B indicates that the B-factors of atoms that surround this water/ion are suspicious. See: swift.cmbi.ru.nl/teach/theory/ for a detailed explanation.

 633 HOH   ( 495 )  N      O  1.13  K  4

Warning: Possible wrong residue type

The residues listed in the table below have a weird environment that cannot be improved by rotamer flips. This can mean one of three things, non of which WHAT CHECK really can do much about. 1) The side chain has actually another rotamer than is present in the PDB file; 2) A counter ion is present in the structure but is not given in the PDB file; 3) The residue actually is another amino acid type. The annotation 'Alt-rotamer' indicates that WHAT CHECK thinks you might want to find an alternate rotamer for this residue. The annotation 'Sym-induced' indicates that WHAT CHECK believes that symmetry contacts might have something to do with the difficulties of this residue's side chain. Determination of these two annotations is difficult, so their absence is less meaningful than their presence. The annotation Ligand-bound indicates that a ligand seems involved with this residue. In nine of ten of these cases this indicates that the ligand is causing the weird situation rather than the residue.

  22 ASP   ( 103-)  N   H-bonding suggests Asn; but Alt-Rotamer
  76 ASP   ( 157-)  N   H-bonding suggests Asn; but Alt-Rotamer
 105 ASP   ( 185-)  N   H-bonding suggests Asn; but Alt-Rotamer
 163 ASP   ( 243-)  N   H-bonding suggests Asn; but Alt-Rotamer
 186 GLU   ( 266-)  N   H-bonding suggests Gln
 405 ASP   (  17-)  L   H-bonding suggests Asn
 479 ASP   (  91-)  L   H-bonding suggests Asn
 563 ASP   (  65-)  H   H-bonding suggests Asn; but Alt-Rotamer

Final summary

Note: Summary report for users of a structure

This is an overall summary of the quality of the structure as compared with current reliable structures. This summary is most useful for biologists seeking a good structure to use for modelling calculations.

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 :  -0.925
  2nd generation packing quality :  -1.679
  Ramachandran plot appearance   :  -3.435 (poor)
  chi-1/chi-2 rotamer normality  :  -4.062 (bad)
  Backbone conformation          :  -1.341

RMS Z-scores, should be close to 1.0:
  Bond lengths                   :   0.558 (tight)
  Bond angles                    :   0.843
  Omega angle restraints         :   0.333 (tight)
  Side chain planarity           :   0.777
  Improper dihedral distribution :   1.203
  Inside/Outside distribution    :   1.018

Note: Summary report for depositors of a structure

This is an overall summary of the quality of the X-ray structure as compared with structures solved at similar resolutions. This summary can be useful for a crystallographer to see if the structure makes the best possible use of the data. Warning. This table works well for structures solved in the resolution range of the structures in the WHAT IF database, which is presently (summer 2008) mainly 1.1 - 1.3 Angstrom. The further the resolution of your file deviates from this range the more meaningless this table becomes.

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 : 2.20


Structure Z-scores, positive is better than average:

  1st generation packing quality :  -0.2
  2nd generation packing quality :  -0.8
  Ramachandran plot appearance   :  -1.9
  chi-1/chi-2 rotamer normality  :  -2.5
  Backbone conformation          :  -1.2

RMS Z-scores, should be close to 1.0:
  Bond lengths                   :   0.558 (tight)
  Bond angles                    :   0.843
  Omega angle restraints         :   0.333 (tight)
  Side chain planarity           :   0.777
  Improper dihedral distribution :   1.203
  Inside/Outside distribution    :   1.018
==============

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.