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All-Atom-Contact Analysis

Once all H atoms have been added and optimized (with Reduce), analysis of atomic overlaps (both clashes and H-bonds) makes a very sensitive and reliable way to find all the problem areas in a model, and often indicates how to fix them. In assessing a deposited crystal structure, the local presence or absence of all-atom clashes is an excellent way to guage the local accuracy of that part of the structure. The method is sensitive because H atom clashes are almost never minimized in refinement. That means, however, that you should not be surprised to find such problems in respectable, well-done structures.

The figure at top left shows the all-atom contacts for a Thr fit backwards in a 1.7 Å structure (not uncommon, since electon density for branched sidechains is sometimes straight and bar-shaped rather than showing the tetrahedral carbon position). Such misfittings result in poor rotamers and characteristic bond-angle distortions that aid in diagnosis. Below are contacts for the same Thr, idealized and refit. For an interactive Java Mage tour of the process of evaluating and fixing clashes in this molecule, follow this link to Clash Report kinemages.

To get a graphics display of the serious clashes (non-Hbond overlaps >= 0.4 Å) in the 3D structure for PDB file 1xyz, run the following commands in Unix or Linux, where you have in your path the current versions of Reduce, Probe, Mage and Prekin (see Software page). This works well even on quite large protein and/or nucleic acid structures.

If you turn off sidechains, het groups, distant and close contacts, small overlaps, and Hbonds (leaving only backbone and serious overlaps >= 0.4 Å), you can easily see the red spikes that indicate problems (as in the figure below).

Pickcenter on each such area in turn, zoom in, and turn on sidechains again to see what is clashing. If there are too many clashes, try running Probe again with the "all" selection replaced by "blt40", to concentrate on the more important, and more fixable, clashes at low B.

If you prefer to work with a list of problem areas (clusters of clashes), download the Unix script clashlistcluster from the scripts download page. First run Reduce as above to add hydrogens, then type:

("Name" is an optional identifier, its text will appear in each clash listed.) This makes a plain text file you can read or print out. All clashes of 0.4 Å or more are listed, grouped into local clusters; e.g., the Thr shown above has seven seriously overlapping pairs that are all part of one local problem.

Asn / Gln / His Flips

Addition of all-atom clash information to H-bond analysis allows clear resolution of the 180° orientation ambiguities of sidechain amides or imidazoles in almost all cases, as described in Word et al. (1999) J. Mol. Biol. 285: 1735. [abstract] The figure shows the correct (green) and incorrect (pink) flip orientations for a doubly H-bonded Gln-Asn pair, which have equivalent H-bonds in the two states but are easily assigned because of the physically impossible overlap of an epsilon-NH and the C-alpha H in the lower panel. To see this same example interactively in Java Mage, follow this link to the Asn / Gln flip kinemage in the Java Mage section. (Your browser must be Java enabled.)

Reduce will do these corrections automatically if run with the -build flag:

producing a modified and commented PDB file with H atoms added and optimized, in this case including the optimization of Asn/Gln/His flips. USERMOD records in the file header tell you which residues were flipped and give their score differences.

If you want to evaluate Reduce's automated decisions (which we would recommend unless you are doing high-throughput structural genomics), a script is available to produce illustrative graphics output - either for Asn/Gln flips or for His flips. (The two cases must be shown separately, because NQ flips exchange donors and acceptors but His flips also exchange H-bonding and non-Hbonding groups.)

To set up for this, download the Perl script "flipkin" from the Software page. Put it in a directory in your path, and make sure it is executable. The script expects to find Perl in the /usr/sbin directory; if it is somewhere else then edit the first line of the script to reflect that. [Note: If you don't have Perl, you can do this process with a package of shell scripts available as a tar file from the Software page.]

First run Reduce:

Then, using both the original and the Reduce-modified PDB files, create the animated kinemage files with a view for each relevant sidechain:

Look at these kinemages in Mage, animating between contact displays for the two alternatives at each sidechain and paying particular attention to the ones Reduce flipped (marked with * on the View pulldown menu).

Note that the local H-bond network has been separately optimized for each flip alternative. Also, Reduce in build mode applies a penalty to keep it from flipping when the two scores are nearly equal; you can change that penalty with the -pen flag, e.g. omitting it ( -pen0 ) or increasing it ( -pen1.5 ). About 85% of Asn/Gln orientations can be unambiguously assigned by this method; the rest are highly exposed and propably populate both states.

Ramachandran Redux

Please see the linked pdf pre-press document for new contours and related information.

Improved definitions are given here for the "Preferred" versus "Allowed but Disfavored" versus "Forbidden" regions of Phi-Psi space obtained from a large, accurate, B-factor filtered database (500 selected proteins at 1.8 Šresolution or better, residues with backbone B values less than 30 Ų: about 100,000 data points)

This figure shows the phi, psi distribution of data points for the non-Gly, non-Pro general case, with the preferred (core) region outlined in orange and the disfavored but allowed region outlined in gold. This new data shows a core region (98% of the data) that almost exactly matches the "strict" single region defined by Kleywegt & Jones (1996, Structure, 4, 1395), but it has in common with ProCheck (Laskowski et al., 1993, J. Appl. Crystallogr. 26, 283) the definition of an allowed outer region. However, it is now clear that the early ProCheck regions were too permissive in many places and missed the now-quite-distinct gamma-turn region near +70°, -60°.

A kinemage file Rama500noGPc.kin of the above distribution and outlines is available. When viewed in the Mage display program, any data point can be clicked on to find its residue number and PDB code, and, if the "X Y Z point" tool is active, its phi-psi values as well. The kinemage also includes the high-B data, to show its greater scatter.

Although peak heights and shapes differ among the 18 non-Gly, non-Pro amino acids, the outline of the preferred and allowed regions are remarkably consistent. However, Gly and Pro are each very different, and using their individual distributions for structure validation is very desirable.

Gly, in spite of its greater flexibility, still occupies only about half the area of the Ramachandran plot and is the residue which phi,psi values are most prone to error (since it has fewer observable atoms.) A twofold-symmetrized version of the Gly distribution is shown at left.

A kinemage file ( Rama500Glyc.kin) can be downloaded to view in Mage. It shows the non-symmetrized Gly data points, which fit the symmetrized region boundaries well but have more points with positive phi because they are more uniquely useful to a protein.

The closed ring of Pro makes it the most constrained residue type, with Phi values between about -50° and -90°. In addition to the expected peaks in the helical region and the poly-Pro region of beta, the Pro distribution at left shows a minor peak in between them, which corresponds to the inverse gamma-turn region.

This distribution includes both trans and cis Pro although the differences are substantial, with cis Pro confined to the lower phi values. A kinemage file ( Rama500Proc.kin) may be downloaded for viewing in Mage; it allows animating between the cis and trans data.

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