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.
67 ARG ( 86-) A - CG 67 ARG ( 86-) A - CD 67 ARG ( 86-) A - NE 67 ARG ( 86-) A - CZ 67 ARG ( 86-) A - NH1 67 ARG ( 86-) A - NH2 80 PRO ( 95K) A - CD
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.
79 ILE ( 95C) 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'.
80 PRO ( 95-) A CG
In X-ray the coordinates must be located in density. Mobility or disorder sometimes cause this density to be so poor that the positions of the atoms cannot be determined. Crystallographers tend to leave out the atoms in such cases. In many cases the N- or C-terminal residues are too disordered to see. In case of the N-terminus, you can see from the residue numbers if there are missing residues, but at the C-terminus this is impossible. Therefore, often the position of the backbone nitrogen of the first residue missing at the C-terminal end is calculated and added to indicate that there are missing residues. As a single N causes validation trouble, we remove these single-N-residues before doing the validation. But, if you get weird errors at, or near, the left-over incomplete C-terminal residue, please check by hand if a missing Oxt or removed N is the cause.
79 ILE ( 95-) A
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 : 7.35
Note: B-factor plot
The average atomic B-factor per residue is plotted as function of the residue
Chain identifier: A
Nomenclature related problems
Warning: Tyrosine convention problem
The tyrosine residues listed in the table below have their chi-2 not between
-90.0 and 90.0
5 TYR ( 20-) A 41 TYR ( 59-) A 45 TYR ( 64-) A 75 TYR ( 94-) A 154 TYR ( 172-) A 209 TYR ( 228-) A
60 PHE ( 79-) A 215 PHE ( 234-) A
30 ASP ( 48-) A 57 ASP ( 77-) A 97 ASP ( 113-) A
94 GLU ( 110-) A 130 GLU ( 148-) A 169 GLU ( 186-) A
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.
25 GLY ( 43-) A CA C 1.58 4.5 117 THR ( 135-) A CB OG1 1.51 4.8 181 GLY ( 197-) A N CA 1.52 4.0
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
| 0.994523 0.000764 0.000831| | 0.000764 0.993532 0.001225| | 0.000831 0.001225 0.995182|Proposed new scale matrix
| 0.020698 -0.000016 -0.000017| | -0.000016 0.020719 -0.000025| | -0.000004 -0.000006 0.005019|With corresponding cell
A = 48.313 B = 48.265 C = 199.236 Alpha= 89.859 Beta= 89.904 Gamma= 89.912
The CRYST1 cell dimensions
A = 48.580 B = 48.580 C = 200.200 Alpha= 90.000 Beta= 90.000 Gamma= 90.000
(Under-)estimated Z-score: 11.308
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.
20 ASN ( 35-) A -C N CA 129.23 4.2 20 ASN ( 35-) A CA CB CG 117.90 5.3 21 GLU ( 39-) A -C N CA 114.38 -4.1 24 CYS ( 42-) A N CA CB 117.43 4.1 25 GLY ( 43-) A N CA C 100.09 -4.3 30 ASP ( 48-) A CA CB CG 117.53 4.9 31 PRO ( 49-) A -O -C N 127.83 4.2 32 SER ( 50-) A CA CB OG 101.81 -4.6 39 HIS ( 57-) A CG ND1 CE1 109.74 4.1 46 GLN ( 65-) A N CA CB 118.72 4.8 51 ARG ( 70-) A CD NE CZ 129.92 4.6 53 ASN ( 72-) A CA CB CG 107.15 -5.4 55 PHE ( 74-) A CA CB CG 118.10 4.3 72 HIS ( 91-) A CA CB CG 107.63 -6.2 77 PRO ( 95-) A -O -C N 129.30 5.2 79 PRO ( 95-) A N CA CB 112.11 8.3 80 VAL ( 96-) A CA C O 129.26 5.0 94 GLU ( 110-) A CA CB CG 122.79 4.3 97 ASP ( 113-) A N CA CB 118.85 4.9 97 ASP ( 113-) A CA CB CG 116.85 4.2 107 LEU ( 123-) A C CA CB 120.53 5.5 117 THR ( 135-) A CA CB OG1 101.43 -5.4 120 ALA ( 138-) A -O -C N 129.51 4.1 123 TRP ( 141-) A -O -C N 130.34 4.6 126 THR ( 144-) A -C N CA 129.41 4.3 126 THR ( 144-) A CA CB OG1 101.80 -5.2 127 ASN ( 145-) A CA CB CG 107.01 -5.6 130 GLU ( 148-) A CA CB CG 127.46 6.7 133 VAL ( 151-) A -O -C N 129.45 4.0 135 HIS ( 153-) A CA CB CG 108.60 -5.2 148 GLU ( 166-) A CA CB CG 124.17 5.0 172 LYS ( 188-) A CG CD CE 123.75 5.4 175 CYS ( 191-) A C CA CB 118.52 4.4 176 ALA ( 192-) A N CA CB 103.17 -4.8 176 ALA ( 192-) A C CA CB 117.62 4.7 178 ASP ( 194-) A CA C O 112.83 -4.7 178 ASP ( 194-) A CA CB CG 116.92 4.3 213 ILE ( 232-) A C CA CB 120.77 5.6 227 PRO ( 246-) A CA C O 113.93 -4.0
30 ASP ( 48-) A 57 ASP ( 77-) A 94 GLU ( 110-) A 97 ASP ( 113-) A 130 GLU ( 148-) A 169 GLU ( 186-) A
25 GLY ( 43-) A 4.95 78 LEU ( 95-) A 4.24 161 VAL ( 179-) A 4.14
Tau angle RMS Z-score : 1.515
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.
209 TYR ( 228-) A 4.25
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.
36 THR ( 54-) A -2.9 204 LYS ( 223-) A -2.8 55 PHE ( 74-) A -2.7 213 ILE ( 232-) A -2.4 20 ASN ( 35-) A -2.3 87 LEU ( 103-) A -2.3 103 LYS ( 119-) A -2.3 205 THR ( 224-) A -2.3 220 LYS ( 239-) A -2.3 35 ILE ( 53-) A -2.3 51 ARG ( 70-) A -2.2 68 GLN ( 87-) A -2.1 130 GLU ( 148-) A -2.1 94 GLU ( 110-) A -2.1 196 GLY ( 216-) A -2.1 12 GLN ( 27-) 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.
20 ASN ( 35-) A Poor phi/psi 85 ASN ( 101-) A Poor phi/psi 101 GLY ( 117-) A Poor phi/psi 156 ASP ( 174-) A Poor phi/psi 194 SER ( 214-) A Poor phi/psi 198 THR ( 218-) A PRO omega poor 201 ALA ( 221-) A Poor phi/psi 204 LYS ( 223-) A Poor phi/psi chi-1/chi-2 correlation Z-score : -4.859
chi-1/chi-2 correlation Z-score : -4.859
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.
140 VAL ( 158-) A 0.38
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!
5 TYR ( 20-) A 0 9 LYS ( 24-) A 0 10 ASN ( 25-) A 0 11 SER ( 26-) A 0 12 GLN ( 27-) A 0 14 TRP ( 29-) A 0 19 ILE ( 34-) A 0 20 ASN ( 35-) A 0 21 GLU ( 39-) A 0 22 TYR ( 40-) A 0 32 SER ( 50-) A 0 40 CYS ( 58-) A 0 41 TYR ( 59-) A 0 42 SER ( 60-) A 0 43 ASN ( 61-) A 0 44 ASN ( 63-) A 0 49 LEU ( 68-) A 0 51 ARG ( 70-) A 0 55 PHE ( 74-) A 0 57 ASP ( 77-) A 0 61 ALA ( 80-) A 0 72 HIS ( 91-) A 0 77 PRO ( 95-) A 0 78 LEU ( 95-) A 0 79 PRO ( 95-) A 0And so on for a total of 106 lines.
Standard deviation of omega values : 3.151
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!
196 GLY ( 216-) A 1.77 21
59 PRO ( 79-) A 0.15 LOW 79 PRO ( 95-) A 0.00 LOW 203 PRO ( 222-) A 0.05 LOW
73 PRO ( 92-) A -120.3 half-chair C-delta/C-gamma (-126 degrees) 182 PRO ( 198-) A -50.1 half-chair C-beta/C-alpha (-54 degrees) 199 PRO ( 219-) A -57.4 half-chair C-beta/C-alpha (-54 degrees) 206 PRO ( 225-) A 27.1 envelop C-delta (36 degrees)
The contact distances of all atom pairs have been checked. Two atoms are said to `bump' if they are closer than the sum of their Van der Waals radii minus 0.40 Angstrom. For hydrogen bonded pairs a tolerance of 0.55 Angstrom is used. The first number in the table tells you how much shorter that specific contact is than the acceptable limit. The second distance is the distance between the centres of the two atoms. Although we believe that two water atoms at 2.4 A distance are too close, we only report water pairs that are closer than this rather short distance.
The last text-item on each line represents the status of the atom pair. If the final column contains the text 'HB', the bump criterion was relaxed because there could be a hydrogen bond. Similarly relaxed criteria are used for 1-3 and 1-4 interactions (listed as 'B2' and 'B3', respectively). BL indicates that the B-factors of the clashing atoms have a low B-factor thereby making this clash even more worrisome. INTRA and INTER indicate whether the clashes are between atoms in the same asymmetric unit, or atoms in symmetry related asymmetric units, respectively.
143 HIS ( 161-) A ND1 <-> 167 GLU ( 185-) A OE2 0.36 2.34 INTRA BL 51 ARG ( 70-) A NH2 <-> 56 LYS ( 75-) A O 0.31 2.39 INTRA 168 MET ( 186-) A O <-> 204 LYS ( 223-) A N 0.25 2.45 INTRA 113 LYS ( 131-) A NZ <-> 230 HOH ( 308 ) A O 0.24 2.46 INTRA 113 LYS ( 131-) A CD <-> 230 HOH ( 373 ) A O 0.23 2.57 INTRA 10 ASN ( 25-) A ND2 <-> 230 HOH ( 363 ) A O 0.23 2.47 INTRA 7 CYS ( 22-) A SG <-> 138 GLN ( 156-) A C 0.20 3.20 INTRA BL 127 ASN ( 145-) A ND2 <-> 230 HOH ( 316 ) A O 0.20 2.50 INTRA 115 GLY ( 133-) A N <-> 144 LEU ( 162-) A O 0.18 2.52 INTRA BL 87 LEU ( 103-) A CD1 <-> 210 ALA ( 229-) A CB 0.16 3.04 INTRA BL 5 TYR ( 20-) A O <-> 139 CYS ( 157-) A N 0.16 2.54 INTRA BL 55 PHE ( 74-) A CE1 <-> 135 HIS ( 153-) A CG 0.16 3.04 INTRA 157 ASN ( 175-) A ND2 <-> 230 HOH ( 370 ) A O 0.15 2.55 INTRA BF 67 ARG ( 86-) A NH1 <-> 230 HOH ( 364 ) A O 0.15 2.55 INTRA BL 186 ASP ( 202-) A OD2 <-> 230 HOH ( 290 ) A O 0.14 2.26 INTRA 30 ASP ( 48-) A C <-> 32 SER ( 50-) A N 0.14 2.76 INTRA BL 55 PHE ( 74-) A CZ <-> 135 HIS ( 153-) A CE1 0.13 3.07 INTRA 216 THR ( 235-) A O <-> 220 LYS ( 239-) A N 0.13 2.57 INTRA BL 50 GLY ( 69-) A N <-> 102 VAL ( 118-) A CG2 0.12 2.98 INTRA BL 118 CYS ( 136-) A O <-> 142 ILE ( 160-) A N 0.11 2.59 INTRA BL 52 ASN ( 71-) A OD1 <-> 230 HOH ( 309 ) A O 0.11 2.29 INTRA 156 ASP ( 174-) A CB <-> 157 ASN ( 175-) A N 0.11 2.59 INTRA B3 222 VAL ( 241-) A O <-> 226 ASN ( 245-) A ND2 0.11 2.59 INTRA 118 CYS ( 136-) A N <-> 142 ILE ( 160-) A O 0.10 2.60 INTRA BL 218 TRP ( 237-) A NE1 <-> 230 HOH ( 345 ) A O 0.10 2.60 INTRAAnd so on for a total of 66 lines.
Chain identifier: A
Warning: Abnormal packing environment for some residues
The residues listed in the table below have an unusual packing environment.
The packing environment of the residues is compared with the average packing environment for all residues of the same type in good PDB files. A low packing score can indicate one of several things: Poor packing, misthreading of the sequence through the density, crystal contacts, contacts with a co-factor, or the residue is part of the active site. It is not uncommon to see a few of these, but in any case this requires further inspection of the residue.
204 LYS ( 223-) A -5.68 110 LYS ( 128-) A -5.30 67 ARG ( 86-) A -5.17 169 GLU ( 186-) A -5.08
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.
22 TYR ( 40-) A -2.77
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.
230 HOH ( 366 ) A O 230 HOH ( 375 ) A O 230 HOH ( 381 ) A O 230 HOH ( 384 ) A O Metal-coordinating Histidine residue 39 fixed to 1 Metal-coordinating Histidine residue 81 fixed to 1 Metal-coordinating Histidine residue 83 fixed to 1
10 ASN ( 25-) A
Hydrogen bond donors that are buried inside the protein normally use all of their hydrogens to form hydrogen bonds within the protein. If there are any non hydrogen bonded buried hydrogen bond donors in the structure they will be listed here. In very good structures the number of listed atoms will tend to zero.
Waters are not listed by this option.
14 TRP ( 29-) A N 61 ALA ( 80-) A N 110 LYS ( 128-) A N 121 SER ( 139-) A N 169 GLU ( 186-) A N 172 LYS ( 188-) A N 179 SER ( 195-) A N Only metal coordination for 39 HIS ( 57-) A NE2 Only metal coordination for 81 HIS ( 97-) A NE2
The second part of the table mostly gives an impression of how well the model conforms to common refinement restraint values. The first part of the table shows a number of global quality indicators.
Structure Z-scores, positive is better than average:
1st generation packing quality : 0.811 2nd generation packing quality : -0.189 Ramachandran plot appearance : -0.931 chi-1/chi-2 rotamer normality : -4.859 (bad) Backbone conformation : -0.421
Bond lengths : 0.962 Bond angles : 1.566 Omega angle restraints : 0.573 (tight) Side chain planarity : 1.533 Improper dihedral distribution : 1.481 Inside/Outside distribution : 1.000
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 : 1.80
Structure Z-scores, positive is better than average:
1st generation packing quality : 1.2 2nd generation packing quality : -0.6 Ramachandran plot appearance : -0.9 chi-1/chi-2 rotamer normality : -4.2 (bad) Backbone conformation : -0.7
Bond lengths : 0.962 Bond angles : 1.566 Omega angle restraints : 0.573 (tight) Side chain planarity : 1.533 Improper dihedral distribution : 1.481 Inside/Outside distribution : 1.000 ==============
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.