************************************************************************ ********** REPORT OF PROTEIN ANALYSIS by the WHAT IF program ********** ************************************************************************ Date : 2012-05-26 This report was created by WHAT IF version 20120321-1556 This document is a WHAT_CHECK-report that holds the findings of the WHAT IF program during the analysis of a PDB-file. Each reported fact has an assigned severity, one of: error : Items marked as errors are considered severe problems requiring immediate attention. warning: Either less severe problems or uncommon structural features. These still need special attention. note : Statistical values, plots, or other verbose results of tests and analyses that have been performed. If alternate conformations are present, only the first is evaluated. Hydrogen atoms are only included if explicitly requested, and even then they are not used in all checks. The software functions less well for non-canonical amino acids and exotic ligands than for the 20 canonical residues and canonical nucleic acids. Some remarks regarding the output: Residues/atoms in tables are normally given in a few parts: A number. This is the internal sequence number of the residue used by WHAT IF. The first residues in the file get number 1, 2, etc. The residue type. Normally this is a three letter amino acid type. The sequence number, between brackets. This is the residue number as it was given in the input file. It can be followed by the insertion code. The chain identifier. A single character. If no chain identifier was given in the input file, this will be a minus sign or a blank. A model number. If no model number exists, like in most X-ray files, this will be a blank or occasionally a minus sign. In case an atom is part of the output, the atom will be listed using the PDB nomenclature for type and identifier. To indicate the normality of a score, the score may be expressed as a Z-value or Z-score. This is just the number of standard deviations that the score deviates from the expected value. A property of Z-values is that the root-mean-square of a group of Z-values (the RMS Z-value) is expected to be 1.0. Z-values above 4.0 and below $-4.0$ are very uncommon. If a Z-score is used in WHAT IF, the accompanying text will explain how the expected value and standard deviation were obtained. The names of nucleic acids are DGUA, DTHY, OCYT, OADE, etc. The first character is a D or O for DNA or RNA respectively. This circumvents ambiguities in the many old PDB files in which DNA and RNA were both called A, C, G, and T. ERROR. C1A ( C1A) does not belong in NAG ( 130) A 0 ERROR. C2A ( C2A) does not belong in NAG ( 130) A 0 ERROR. C3A ( C3A) does not belong in NAG ( 130) A 0 ERROR. C4A ( C4A) does not belong in NAG ( 130) A 0 ERROR. C5A ( C5A) does not belong in NAG ( 130) A 0 ERROR. C6A ( C6A) does not belong in NAG ( 130) A 0 ERROR. C7A ( C7A) does not belong in NAG ( 130) A 0 ERROR. C8A ( C8A) does not belong in NAG ( 130) A 0 ERROR. N2A ( N2A) does not belong in NAG ( 130) A 0 ERROR. O3A ( O3A) does not belong in NAG ( 130) A 0 ERROR. O4A ( O4A) does not belong in NAG ( 130) A 0 ERROR. O5A ( O5A) does not belong in NAG ( 130) A 0 ERROR. O6A ( O6A) does not belong in NAG ( 130) A 0 ERROR. O7A ( O7A) does not belong in NAG ( 130) A 0 ERROR. C1B ( C1B) does not belong in NAG ( 130) A 0 ERROR. C2B ( C2B) does not belong in NAG ( 130) A 0 ERROR. C3B ( C3B) does not belong in NAG ( 130) A 0 ERROR. C4B ( C4B) does not belong in NAG ( 130) A 0 ERROR. C5B ( C5B) does not belong in NAG ( 130) A 0 ERROR. C6B ( C6B) does not belong in NAG ( 130) A 0 ERROR. C7B ( C7B) does not belong in NAG ( 130) A 0 ERROR. C8B ( C8B) does not belong in NAG ( 130) A 0 ERROR. N2B ( N2B) does not belong in NAG ( 130) A 0 ERROR. O3B ( O3B) does not belong in NAG ( 130) A 0 ERROR. O4B ( O4B) does not belong in NAG ( 130) A 0 ERROR. O5B ( O5B) does not belong in NAG ( 130) A 0 ERROR. O6B ( O6B) does not belong in NAG ( 130) A 0 ERROR. O7B ( O7B) does not belong in NAG ( 130) A 0 ERROR. C1C ( C1C) does not belong in NAG ( 130) A 0 ERROR. C2C ( C2C) does not belong in NAG ( 130) A 0 ERROR. C3C ( C3C) does not belong in NAG ( 130) A 0 ERROR. C4C ( C4C) does not belong in NAG ( 130) A 0 ERROR. C5C ( C5C) does not belong in NAG ( 130) A 0 ERROR. C6C ( C6C) does not belong in NAG ( 130) A 0 ERROR. C7C ( C7C) does not belong in NAG ( 130) A 0 ERROR. C8C ( C8C) does not belong in NAG ( 130) A 0 ERROR. OC ( OC ) does not belong in NAG ( 130) A 0 ERROR. O3C ( O3C) does not belong in NAG ( 130) A 0 ERROR. O4C ( O4C) does not belong in NAG ( 130) A 0 ERROR. O6C ( O6C) does not belong in NAG ( 130) A 0 ERROR. O7C ( O7C) does not belong in NAG ( 130) A 0 ERROR. N2C ( N2C) does not belong in NAG ( 130) A 0 ERROR. O1L ( O1L) does not belong in NAG ( 130) A 0 ======================================================================== ==== Compound code /home/whatif/httpd/htdocs/servers/tmp//tmpm3GLCa/====L1.fil ======================================================================== # 1 # Error: Missing unit cell information No SCALE matrix is given in the PDB file. # 2 # Error: Missing symmetry information Problem: No CRYST1 card is given in the PDB file. SYMMETRY will be unavailable for this molecule. # 3 # Note: No strange inter-chain connections detected No covalent bonds have been detected between molecules with non-identical chain identifiers. Id: INTCNEU # 4 # Note: No duplicate atom names in ligands All atom names in ligands (if any) seem adequately unique. # 5 # Note: In all cases the primary alternate atom was used WHAT CHECK saw no need to make any alternate atom corrections (which means they are all correct, or there are none). # 6 # Note: No residues detected inside ligands Either this structure does not contain ligands with amino acid groups inside it, or their naming is proper (enough). # 7 # Note: No attached groups interfere with hydrogen bond calculations It seems there are no sugars, lipids, etc., bound (or very close) to atoms that otherwise could form hydrogen bonds. # 8 # Note: No probable side chain atoms with zero occupancy detected. Either there are no side chain atoms with zero occupancy, or the side chain atoms with zero occupancy were not present in the input PDB file (in which case they are listed as missing atoms), or their positions are sufficiently improbable to warrant a zero occupancy. # 9 # Note: No probable backbone atoms with zero occupancy detected. Either there are no backbone atoms with zero occupancy, or the backbone atoms with zero occupancy were not present in the input PDB file (in which case they are listed as missing atoms), or their positions are sufficiently improbable to warrant a zero occupancy. # 10 # Note: All residues have a complete backbone. No residues have missing backbone atoms. # 11 # Note: No C-alpha only residues There are no residues that consist of only an alpha carbon atom. # 12 # Note: Non-canonical residues WHAT CHECK has not detected any non-canonical residue(s). # 13 # Note: Content of the PDB file as interpreted by WHAT CHECK Content of the PDB file as interpreted by WHAT CHECK. WHAT CHECK has read your PDB file, and stored it internally in what is called 'the soup'. The content of this soup is listed here. An extensive explanation of all frequently used WHAT CHECK output formats can be found at swift.cmbi.ru.nl. Look under output formats. A course on reading this 'Molecules' table is part of the WHAT CHECK website. 1 1 ( 1) 129 ( 129) A Protein checkset 2 130 ( 130) 130 ( 130) A Sugar checkset # 14 # Note: Some notes regarding the PDB file contents The numbers and remarks listed below have no explicit validation purpose, they are merely meant for the crystallographer or NMR spectroscopists to perhaps pinpoint something unexpected. See the WHAT CHECK course [REF] for an explanation of terms like 'poor', 'missing', etcetera (in case those words pop up in the lines underneath this message). The total number of amino acids found is 129. Number of (recognized) sugars: 1 FMT1551O # 15 # Note: Secondary structure This is the secondary structure according to DSSP. Only helix (H), overwound or 3/10-helix (3), strand (S), turn (T) and coil (blank) are shown [REF]. All DSSP related information can be found at swift.cmbi.ru.nl/gv/dssp/ This is not really a structure validation option, but a very scattered secondary structure (i.e. many strands of only a few residues length, many Ts inside helices, etc) tends to indicate a poor structure. A full explanation of the DSSP secondary structure determination program together with a series of examples can be found at the WHAT CHECK website [REF]. Secondary structure assignment 10 20 30 40 50 60 | | | | | | 1 - 60 KVYDRCELARALKASGMDGYAGNSLPNWVCLSKWESSYNTQATNRNTDGSTDYGIFQINS ( 1)-( 60) HHHHHHHHHHTT TT TT HHHHHHHHHHHHTT TT SSS TTT SSSTTTTSST 70 80 90 100 110 120 | | | | | | 61 - 120 RYWCDDGRTPGAKNVCGIRCSQLLTDDLTVAIRCAKRVVLDPNGIGAWVAWRLHCQNQDL ( 61)-( 120)TTT T TT TT T 3333TTTT HHHHHHHHHHTTTTT3333 HHHHHHTTTT 121 - 129 RSYVAGCGV ( 121)-( 129)333TTTT # 16 # Note: No rounded coordinates detected No significant rounding of atom coordinates has been detected. # 17 # Note: No artificial side chains detected No artificial side-chain positions characterized by chi-1=0.0 or chi-1=180.0 have been detected. # 18 # Warning: Unexpected atoms encountered While reading the PDB file, at least one atom was encountered that was not expected in the residue. This might be caused by a naming convention problem. It can also mean that a residue was found protonated that normally is not (e.g. aspartic acid). The unexpected atoms have been discarded; in case protons were deleted that actually might be needed, they will later be put back by the hydrogen bond validation software. This normally is not a warning you should worry too much about. # 19 # 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'. 130 NAG ( 130-) A O4 130 NAG ( 130-) A C4 130 NAG ( 130-) A C1 130 NAG ( 130-) A O5 130 NAG ( 130-) A C5 130 NAG ( 130-) A C6 130 NAG ( 130-) A O6 130 NAG ( 130-) A C3 130 NAG ( 130-) A O3 130 NAG ( 130-) A C2 130 NAG ( 130-) A N2 130 NAG ( 130-) A C7 130 NAG ( 130-) A O7 130 NAG ( 130-) A C8 # 20 # Note: All B-factors fall in the range 0.0 - 100.0 All B-factors are larger than zero, and none are observed above 100.0. # 21 # Note: No C-terminal nitrogen detected The PDB indicates that a residue is not the true C-terminus by including only the backbone N of the next residue. This has not been observed in this PDB file. # 22 # Note: C-terminus capping The residues listed in the table below either are pseudo C-terminal residues, or have two groups attached of which neither is the normal C-terminal O. In this table REAL means that the C-terminal residue is likely to be the real C-terminus of its chain; OX means that an incorrect second oxygen (OXT) was detected that should not be there; -O indicates that the 'normal' oxygen (i.e. not the OXT) is missing; OT indicates the detection of any other capping group. C-terminal nitrogen atoms, if any, have already been dealt with in a previous check and are indicated here by -N. PSEUDO means that this is the last visible residue in the chain, but not the real C-terminus, i.e. all residues after this one are missing in this chain. BREAK means that this is the last residue before a chain-break, i.e. the chain continues but after this residue a number of residues is missing. In case a break is observed the number of residues that seems to be missing is shown in brackets. OK means that given the status (REAL, PSEUDO, BREAK), no problems were found. Be aware that we cannot easily see the difference between these errors and errors in the chain and residue numbering schemes. So do not blindly trust the table below. 129 VAL ( 129-) A : REAL Oxt missing # 23 # Note: No OXT found in the middle of chains No OXT groups were found in the middle of protein chains. # 24 # Note: Introduction to the nomenclature section. Nomenclature problems seem, at first, rather unimportant. After all who cares if we call the delta atoms in leucine delta2 and delta1 rather than the other way around. Chemically speaking that is correct. But structures have not been solved and deposited just for chemists to look at them. Most times a structure is used, it is by software in a bioinformatics lab. And if they compare structures in which the one used C delta1 and delta2 and the other uses C delta2 and delta1, then that comparison will fail. Also, we recalculate all structures every so many years to make sure that everybody always can get access to the best coordinates that can be obtained from the (your?) experimental data. These recalculations will be troublesome if there are nomenclature problems. Several nomenclature problems actually are worse than that. At the WHAT CHECK website [REF] you can get an overview of the importance of all nomenclature problems that we list. # 25 # Note: Valine nomenclature OK No errors were detected in valine nomenclature. # 26 # Note: Threonine nomenclature OK No errors were detected in threonine nomenclature. # 27 # Note: Isoleucine nomenclature OK No errors were detected in isoleucine nomenclature. # 28 # Note: Leucine nomenclature OK No errors were detected in leucine nomenclature. # 29 # Note: Arginine nomenclature OK No errors were detected in arginine nomenclature. # 30 # Note: Tyrosine torsion conventions OK No errors were detected in tyrosine torsion angle conventions. # 31 # Note: Phenylalanine torsion conventions OK No errors were detected in phenylalanine torsion angle conventions. # 32 # Note: Aspartic acid torsion conventions OK No errors were detected in aspartic acid torsion angle conventions. # 33 # Note: Glutamic acid torsion conventions OK No errors were detected in glutamic acid torsion angle conventions. # 34 # Note: Phosphate group names OK in DNA/RNA No errors were detected in nucleic acid phosphate group naming conventions. # 35 # Note: Heavy atom naming OK No errors were detected in the atom names for non-hydrogen atoms. Please be aware that the PDB wants us to deliberately make some nomenclature errors; especially in non-canonical amino acids. # 36 # Note: All bond lengths OK All bond lengths are in agreement with standard bond lengths using a tolerance of 4 sigma (both standard values and sigma for amino acids have been taken from Engh and Huber [REF], for DNA/RNA from Parkinson et al [REF]). # 37 # Note: Normal bond length variability Bond lengths were found to deviate normally from the standard bond lengths (values for Protein residues were taken from Engh and Huber [REF], for DNA/RNA from Parkinson et al [REF]). RMS Z-score for bond lengths: 0.789 RMS-deviation in bond distances: 0.017 # 38 # 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. 1 LYS ( 1-) A CG CD CE 122.20 4.7 4 ASP ( 4-) A -C N CA 131.44 5.4 5 ARG ( 5-) A CD NE CZ 129.35 4.3 10 ARG ( 10-) A CA CB CG 104.11 -5.0 27 ASN ( 27-) A CA CB CG 104.27 -8.3 34 TRP ( 34-) A CB CG CD1 132.92 4.0 39 ASN ( 39-) A ND2 CG OD1 116.73 -5.9 47 THR ( 47-) A CA CB OG1 103.22 -4.3 61 ARG ( 61-) A CA CB CG 103.13 -5.5 61 ARG ( 61-) A CB CG CD 104.18 -5.0 63 TRP ( 63-) A CA C O 112.25 -5.0 65 ASP ( 65-) A N CA CB 102.19 -4.9 65 ASP ( 65-) A CA CB CG 108.04 -4.6 69 THR ( 69-) A CA CB OG1 102.42 -4.8 70 PRO ( 70-) A -O -C N 128.74 4.8 79 ARG ( 79-) A CA C O 112.87 -4.7 79 ARG ( 79-) A CB CG CD 105.30 -4.4 86 ASP ( 86-) A C CA CB 118.82 4.6 86 ASP ( 86-) A CA CB CG 118.19 5.6 89 THR ( 89-) A CA CB OG1 103.31 -4.2 98 VAL ( 98-) A CA C O 113.76 -4.1 99 VAL ( 99-) A N CA CB 118.71 4.8 99 VAL ( 99-) A C CA CB 101.31 -4.6 99 VAL ( 99-) A CA CB CG1 103.50 -4.1 100 LEU ( 100-) A CA C O 112.29 -5.0 110 ALA ( 110-) A CA C O 111.92 -5.2 112 ARG ( 112-) A CD NE CZ 130.42 4.8 114 HIS ( 114-) A CA CB CG 108.23 -5.6 114 HIS ( 114-) A ND1 CE1 NE2 106.47 -4.0 114 HIS ( 114-) A CE1 NE2 CD2 112.68 4.4 118 GLN ( 118-) A CG CD NE2 109.87 -4.4 118 GLN ( 118-) A NE2 CD OE1 128.51 5.9 119 ASP ( 119-) A C CA CB 119.01 4.7 119 ASP ( 119-) A CA CB CG 108.45 -4.2 120 LEU ( 120-) A N CA CB 103.04 -4.4 121 ARG ( 121-) A CG CD NE 118.98 5.0 121 ARG ( 121-) A CD NE CZ 133.83 6.7 129 VAL ( 129-) A -C N CA 131.13 5.2 129 VAL ( 129-) A CA C O 110.16 -6.3 # 39 # Note: Normal bond angle variability Bond angles were found to deviate normally from the mean standard bond angles (normal values for protein residues were taken from Engh and Huber [REF], for DNA/RNA from Parkinson et al [REF]). The RMS Z-score given below is expected to be near 1.0 for a normally restrained data set, and this is indeed observed for very high resolution X-ray structures. RMS Z-score for bond angles: 1.759 RMS-deviation in bond angles: 3.216 # 40 # Note: Residue hand check OK No atoms are observed that have the wrong handedness. Be aware, though, that WHAT CHECK might have corrected the handedness of some atoms already. The handedness has not been corrected for any case where the problem is worse than just an administrative discomfort. # 41 # Note: Chirality OK All protein atoms have proper chirality, or there is no intact protein present in the PDB file. The average deviation= 1.307 # 42 # Note: Improper dihedral angle distribution OK The RMS Z-score for all improper dihedrals in the structure is within normal ranges. Improper dihedral RMS Z-score : 1.055 # 43 # 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. 114 HIS ( 114-) A 4.84 53 TYR ( 53-) A 4.02 # 44 # 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 : 1.500 # 45 # Error: Side chain planarity problems The side chains of the residues listed in the table below contain a planar group that was found to deviate from planarity by more than 4.0 times the expected value. For an amino acid residue that has a side chain with a planar group, the RMS deviation of the atoms to a least squares plane was determined. The number in the table is the number of standard deviations this RMS value deviates from the expected value. Not knowing better yet, we assume that planarity of the groups analyzed should be perfect. 61 ARG ( 61-) A 4.61 # 46 # Note: Atoms connected to aromatic rings OK All of the atoms that are connected to planar aromatic rings in side chains of amino-acid residues are in the plane within expected RMS deviations. Since there is no DNA and no protein with hydrogens, no uncalibrated planarity check was performed. Ramachandran Z-score : -0.965 # 47 # Note: Ramachandran Z-score OK The score expressing how well the backbone conformations of all residues correspond to the known allowed areas in the Ramachandran plot is within expected ranges for well-refined structures. Ramachandran Z-score : -0.965 # 48 # 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 CHECK 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. 73 LYS ( 73-) A -2.3 37 SER ( 37-) A -2.3 68 ARG ( 68-) A -2.0 38 TYR ( 38-) A -2.0 # 49 # 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. 37 SER ( 37-) A Poor phi/psi 38 TYR ( 38-) A Poor phi/psi 117 ASN ( 117-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -2.300 # 50 # Note: chi-1/chi-2 angle correlation Z-score OK The score expressing how well the chi-1/chi-2 angles of all residues correspond to the populated areas in the database is within expected ranges for well-refined structures. chi-1/chi-2 correlation Z-score : -2.300 # 51 # Warning: Unusual rotamers The residues listed in the table below have a rotamer that is not seen very often in the database of solved protein structures. This option determines for every residue the position specific chi-1 rotamer distribution. Thereafter it verified whether the actual residue in the molecule has the most preferred rotamer or not. If the actual rotamer is the preferred one, the score is 1.0. If the actual rotamer is unique, the score is 0.0. If there are two preferred rotamers, with a population distribution of 3:2 and your rotamer sits in the lesser populated rotamer, the score will be 0.667. No value will be given if insufficient hits are found in the database. It is not necessarily an error if a few residues have rotamer values below 0.3, but careful inspection of all residues with these low values could be worth it. 4 ASP ( 4-) A 0.36 # 52 # 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, especially if a regular DSSP secondary structure (H or S for helix or strand) is indicated! 128 GLY ( 128-) A 0 105 ILE ( 105-) A H 1 17 MET ( 17-) A 2 127 CYS ( 127-) A 2 # 53 # Note: Backbone conformation Z-score OK The backbone conformation analysis gives a score that is normal for well refined protein structures. Backbone conformation Z-score : -0.164 Omega average and std. deviation= 179.870 2.718 Significant deviations from expected 5.5!!! # 54 # 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 : 2.718 # 55 # 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] 102 PRO ( 102-) A 0.49 HIGH # 56 # Note: PRO puckering phases OK Puckering phases for all PRO residues are normal # 57 # Note: Backbone oxygen evaluation OK All residues for which the local backbone conformation could be found in the WHAT CHECK database have a normal backbone oxygen position. # 58 # Note: Peptide bond conformations There are not enough (intact) amino acids in the file to analyse peptide bond conformations. # 59 # 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. 45 ARG ( 45-) A NH1 <-> 68 ARG ( 68-) A NH2 0.66 2.19 INTRA BF 3 TYR ( 3-) A OH <-> 88 LEU ( 88-) A CD1 0.31 2.49 INTRA BL 3 TYR ( 3-) A CZ <-> 88 LEU ( 88-) A CD1 0.27 2.93 INTRA BL 45 ARG ( 45-) A CZ <-> 68 ARG ( 68-) A NH2 0.27 2.83 INTRA BF 46 ASN ( 46-) A N <-> 50 SER ( 50-) A O 0.11 2.59 INTRA 1 LYS ( 1-) A N <-> 86 ASP ( 86-) A OD1 0.10 2.60 INTRA 101 ASP ( 101-) A CB <-> 102 PRO ( 102-) A CD 0.08 3.02 INTRA 25 LEU ( 25-) A N <-> 26 PRO ( 26-) A CD 0.08 2.92 INTRA BL 64 CYS ( 64-) A C <-> 80 CYS ( 80-) A SG 0.03 3.37 INTRA BL 101 ASP ( 101-) A CA <-> 102 PRO ( 102-) A CD 0.03 2.77 INTRA B3 # 60 # Note: Some notes regarding these bumps The bumps have been binned in 5 categories ranging from 'should deal with' till 'must fix'. Additionally, the integrated sum of all bumps, the squared sum of all bumps, and these latter two values normalized by the number of contacts are listed too for comparison purposes between, for example, small and large proteins. Total bump value: 1.934 Total bump value per residue: 0.077 Total number of bumps: 10 Total squared bump value: 0.714 Total number of bumps in the mildest bin: 6 Total number of bumps in the second bin: 3 Total number of bumps in the middle bin: 1 Total number of bumps in the fourth bin: 0 Total number of bumps in the worst bin: 0 # 61 # Note: Inside/Outside residue distribution normal The distribution of residue types over the inside and the outside of the protein is normal. inside/outside RMS Z-score : 0.919 68 ARG ( 68) : -6.926 # 62 # 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. 68 ARG ( 68-) A -6.93 # 63 # 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. 34 TRP ( 34-) A 36 - SER 36- ( A) -4.48 # 64 # Note: Structural average packing environment OK The structural average packing score is within normal ranges. Average for range 1 - 130 : -0.984 Number of ambiguities touching ambiguities: 0 # 65 # 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. 23 ASN ( 23-) A 46 ASN ( 46-) A # 66 # Note: Histidine type assignments For all complete HIS residues in the structure a tentative assignment to HIS-D (protonated on ND1), HIS-E (protonated on NE2), or HIS-H (protonated on both ND1 and NE2, positively charged) is made based on the hydrogen bond network. A second assignment is made based on which of the Engh and Huber [REF] histidine geometries fits best to the structure. In the table below all normal histidine residues are listed. The assignment based on the geometry of the residue is listed first, together with the RMS Z-score for the fit to the Engh and Huber parameters. For all residues where the H-bond assignment is different, the assignment is listed in the last columns, together with its RMS Z-score to the Engh and Huber parameters. As always, the RMS Z-scores should be close to 1.0 if the residues were restrained to the Engh and Huber parameters during refinement, and if enough (high resolution) data is available. Please note that because the differences between the geometries of the different types are small it is possible that the geometric assignment given here does not correspond to the type used in refinement. This is especially true if the RMS Z-scores are much higher than 1.0. If the two assignments differ, or the `geometry' RMS Z-score is high, it is advisable to verify the hydrogen bond assignment, check the HIS type used during the refinement and possibly adjust it. 114 HIS ( 114-) A HIS-H 0.50 HIS-D 0.71 # 67 # 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. 51 THR ( 51-) A OG1 64 CYS ( 64-) A N 74 ASN ( 74-) A N # 68 # Note: Buried hydrogen bond acceptors OK All buried polar side-chain hydrogen bond acceptors are involved in a hydrogen bond in the optimized hydrogen bond network. # 69 # Note: Some notes regarding these donors and acceptors The donors and acceptors have been counted, also as function of their accessibility. The buried donors and acceptors have been binned in five categories ranging from not forming any hydrogen bond till forming a poor till perfect hydrogen bond. Obviously, the buried donors and acceptors with no or just a poor hydrogen bond should be a topic of concern. Total number of donors: 203 of which buried: 88 Total number of acceptors: 199 of which buried: 72 Total number of donor+acceptors: 22 (donor+acceptor is e.g. the Ser Ogamma that can donate and accept) of which buried: 7 Buried donors: 88 without H-bond: 3 essentially without H-bond: 1 with only a very poor H-bond: 1 with a poor H-bond: 1 with a H-bond: 82 Buried acceptors: 72 without H-bond: 13 essentially without H-bond: 0 with only a very poor H-bond: 0 with a poor H-bond: 0 with a H-bond: 59 # 70 # 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. # 71 # Note: No ions (of a type we can validate) in structure Since there are no ions in the structure of a type we can validate, this check will not be executed. Since there are no waters, the water check has been skipped. SOUP contains no water: # 72 # Note: Content of the PDB file as interpreted by WHAT CHECK Content of the PDB file as interpreted by WHAT CHECK. WHAT CHECK has read your PDB file, and stored it internally in what is called 'the soup'. The content of this soup is listed here. An extensive explanation of all frequently used WHAT CHECK output formats can be found at swift.cmbi.ru.nl. Look under output formats. A course on reading this 'Molecules' table is part of the WHAT CHECK website. 1 1 ( 1) 129 ( 129) A Protein checkset 2 130 ( 130) 130 ( 130) A Sugar checkset # 73 # 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 : -1.211 Ramachandran plot appearance : -0.965 chi-1/chi-2 rotamer normality : -2.300 Backbone conformation : -0.164 RMS Z-scores, should be close to 1.0: Bond lengths : 0.789 Bond angles : 1.759 Omega angle restraints : 0.494 (tight) Side chain planarity : 1.318 Improper dihedral distribution : 1.055 Inside/Outside distribution : 0.919 # 74 # Note: Introduction to refinement recommendations First, be aware that the recommendations for crystallographers listed below are produced by a computer program that was written by a guy who got his PhD in NMR... We have tried to convert the messages written in this report into a small set of things you can do with your refinement software to get a better structure. The things you should do first are listed first. And in some cases you should first fix that problem, then refine a bit further, and then run WHAT CHECK again before looking at other problems. If, for example, WHAT CHECK has found a problem with the SCALE and CRYST cards, then you must first fix that problem, refine the structure a bit further, and run WHAT CHECK again because errors in the SCALE and or CRYST card can lead to many problems elsewhere in the validation process. It is also important to keep in mind that WHAT CHECK is software and that it occasionally totally misunderstands what is the cause of a problem. But, if WHAT CHECK lists a problem there normally is a problem albeit that it not always is the actual problem that gets listed. # 75 # Note: No crippling problems detected Some problems can be so crippling that they negatively influence the validity of other validation steps. If such a problem is detected, it must be solved and some further refinemnet must be done before you can continue working with a new WHAT CHECK report. In this file such problems were not detected. You can therefore try to fix as many problems in one go as you want. # 76 # Error: Bumps in your structure Upon analysing the bumps in your structure, WHAT CHECK got a bit worried. Often this means that you have forgotten to lower the occupancy of overlapping ligands, residues, or water molecules. But, whatever is the origin of this problem, you have to analyse it and fix it. # 77 # Note: Bond angle variabilty Z-score high With a resolution of 1.5-2.5 Angstrom, you dont have enough data to warant the bond angle variability that we observed (more than 1.5). So, you better tighten the screws on the bond angle target values a lot. What you are doing is called overrefinement. # 78 # Note: His, Asn, Gln side chain flips. His, Asn, and Gln have an asymmetry in their side chain that is hard to detect unless you have data at much better than 1.0 Angstrom resolution. WHAT CHECK thinks that your structure contains His, Asn, or Gln residues that will make better hydrogen bonds when flipped around their chi-2, chi-2, or chi-3 side chain torsion angle, respectively. You better check these Asn, His, and Gln residues, and if you use a refinement program that includes molecular dynamics, then you must (after the flips were made) refine a bit further before running WHAT CHECK again. # 79 # Warning: Troublesome residues The residues listed in the table below need to be inspected This table is a very rough attempt to sort the residues according to how badly they need your attention. The idea is that when you sit in in front of the graphics screen and study the residues with the electron density present that you improve the structure most by dealing with the top residues in this list first. 68 ARG ( 68-) A 24.00 3 TYR ( 3-) A 16.39 88 LEU ( 88-) A 15.04 114 HIS ( 114-) A 11.74 99 VAL ( 99-) A 10.18 45 ARG ( 45-) A 10.06 86 ASP ( 86-) A 8.99 121 ARG ( 121-) A 8.80 61 ARG ( 61-) A 8.29 118 GLN ( 118-) A 7.70 129 VAL ( 129-) A 7.61 65 ASP ( 65-) A 7.09 79 ARG ( 79-) A 6.80 119 ASP ( 119-) A 6.63 27 ASN ( 27-) A 6.24 69 THR ( 69-) A 5.99 1 LYS ( 1-) A 4.91 39 ASN ( 39-) A 4.41 110 ALA ( 110-) A 3.92 63 TRP ( 63-) A 3.77 100 LEU ( 100-) A 3.75 10 ARG ( 10-) A 3.75 112 ARG ( 112-) A 3.63 46 ASN ( 46-) A 3.47 120 LEU ( 120-) A 3.29 5 ARG ( 5-) A 3.19 47 THR ( 47-) A 3.19 89 THR ( 89-) A 3.14 98 VAL ( 98-) A 3.10 34 TRP ( 34-) A 3.01 4 ASP ( 4-) A 2.71 25 LEU ( 25-) A 2.13 26 PRO ( 26-) A 2.13 23 ASN ( 23-) A 2.00 64 CYS ( 64-) A 1.78 102 PRO ( 102-) A 1.53 101 ASP ( 101-) A 1.49 50 SER ( 50-) A 1.47 128 GLY ( 128-) A 1.31 70 PRO ( 70-) A 1.20 53 TYR ( 53-) A 1.00 ============== 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 information) 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). R.W.W. Hooft, C.Sander and G.Vriend, Objectively judging the quality of a protein structure from a Ramachandran plot CABIOS (1997), 13, 425--430. 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). Tau angle W.G.Touw and G.Vriend On the complexity of Engh and Huber refinement restraints: the angle tau as example. Acta Crystallogr D 66, 1341--1350 (2010). 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. /home/vriend/whatif/dbdata/pdbout2html After running WHAT IF's WHAT CHECK option many things have happened to the data structure that might not be optimal for many other options. FULCHK therefore is a so-called terminal option, i.e. after running the validation option, WHAT IF will restart without any coordinates in the soup; so the molecule you just checked got deleted together with anything else you might have had in the SOUP. Option not found, try:%NO For obvious reasons $ commands are not allowed in WWW scripts. WHAT IF detected a $ in a WWW script. The command will be listed below. If this command contains something that is potentially harmful to your environment, please mail G Vriend (Vriend@cmbi.kun.nl) which $ command was detected, and from where you got this script. Option:$/home/vriend/whatif/dbdata/pdbout2html ERROR. Trying to close non-opened log file ============== 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 information) 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). R.W.W. Hooft, C.Sander and G.Vriend, Objectively judging the quality of a protein structure from a Ramachandran plot CABIOS (1997), 13, 425--430. 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). Tau angle W.G.Touw and G.Vriend On the complexity of Engh and Huber refinement restraints: the angle tau as example. Acta Crystallogr D 66, 1341--1350 (2010). 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.