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Linda Berens
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Background: Athletic training leads to remodelling of both left and right ventricles with increased myocardial mass and cavity dilatation. Whether changes in cardiac strain parameters occur in response to training is less well established. In this study we investigated the relationship in trained athletes between cardiovascular magnetic resonance (CMR) derived strain parameters of cardiac function and fitness.
Methods: Thirty five endurance athletes and 35 age and sex matched controls underwent CMR at 3.0 T including cine imaging in multiple planes and tissue tagging by spatial modulation of magnetization (SPAMM). CMR data were analysed quantitatively reporting circumferential strain and torsion from tagged images and left and right ventricular longitudinal strain from feature tracking of cine images. Athletes performed a maximal ramp-incremental exercise test to determine the lactate threshold (LT) and maximal oxygen uptake (V̇O2max).
Results: LV circumferential strain at all levels, LV twist and torsion, LV late diastolic longitudinal strain rate, RV peak longitudinal strain and RV early and late diastolic longitudinal strain rate were all lower in athletes than controls. On multivariable linear regression only LV torsion (beta = -0.37, P = 0.03) had a significant association with LT. Only RV longitudinal late diastolic strain rate (beta = -0.35, P = 0.03) had a significant association with V̇O2max.
Conclusions: This cohort of endurance athletes had lower LV circumferential strain, LV torsion and biventricular diastolic strain rates than controls. Increased LT, which is a major determinant of performance in endurance athletes, was associated with decreased LV torsion. Further work is needed to understand the mechanisms by which this occurs.
The example used for this tutorial is called Protein Kinase A (3dnd).To get the requisite files open the GUI and set up the tutorial, as described here, called Protein Kinase A (validation).This should setup the main/GUI window.On the right-hand side, under Crystals, click Validation and map-based comparisons and then select Comprehensive Validation (MolProbity).
In the "Comprehensive Validation" window, browse to the tutorial directory that you specified above, and select 3dnd.pdb as the "Input model", 3dnd.mtz as the "Reflections file", and 3dnd.ligands.cif as the "Restraints (CIF)" file.This CIF file defines restraints for ligands in input PDB file.Now you are ready to run the program, select Run from the top menu bar.
When validation has run, click on the "Compare statistics" button.This will launch a tool called "Polygon" (it may take several seconds), which is used to simultaneously compare R-work, R-free, RMSD-bonds, RMSD-angles, Clashscore, and Average B factor.Well-built models will usually have a small, fairly equilateral polygon, whereas larger or significantly asymmetric deviations are indicative of model problems.
As you can see for 3dnd, both RMSD-bonds and RMSD-angles are a bit large, beyond the expected peaks for structures of similar resolution. This could indicate that misfit areas of the structure are causing geometric strain. We will visit some of these areas in the next sections.
Now select the "MolProbity" tab in the Phenix Comprehensive validation window.Under this tab, you will see sub-tabs for "Summary", "Basic geometry", "Protein", and "Clashes".If your model contained RNA, there would also be an "RNA" tab.
Under the "Summary" tab are most of the same overall statistics that you would find when running the MolProbity webserver.These statistics are colored stoplight-style (green, yellow, and red) as a rough guide the severity of a given result.
This tab also displays Rama-Z score validation.This is a whole-model assessment of how realistic the Ramachandran distribution is and guards against overfitting to Ramachandran criteria.This structure has a reasonable Ramachandran distribution, with all its Rama-Z scores falling within +2 to -2.
From the MolProbity tab, click on the "Basic geometry" tab.Here you find a summary of all bond, angle, dihedral, chirality, and planarity outliers.Outliers will be listed in the associated lists, and each item is clickable, which will center in Coot.Note the bond outlier for Ile A 163 - we'll be seeing this residue again later.
Next, go to the "Protein" tab.This section contains validation information for Ramachandran and rotamer outliers, C-beta deviations, recommended Asn/Gln/His sidechain flips (these have NOT already been done for you, as you can tell if you click on His 39 in the flip list to see it in Coot), and non-trans peptide bonds.
If this structure contained any RNA molecules, there would be an additional tab named "RNA".This section contains validation information for RNA nucleotides with covalent geometry outliers (also found in the Basic geometry tab), RNA nucleotides with sugar pucker outliers, and RNA suites with unexpected backbone conformations.
Go to the "Clashes" tab. This section contains a list of all the steric overlaps >0.4 (i.e. "clashes") in the structure, sorted by severity. Scroll down the list and note that almost all the clashes involve at least one Hydrogen atom. Hydrogens form most of the steric contacts in a structure and are crucial to contact analysis! Phenix, MolProbity, and Coot will generally ensure that hydrogens are added when you need them, but make sure to use phenix.reduce to add hydrogens manually if you are working outside these systems.
Refinement programs are excellent at optimizing details, but may struggle with large changes across energy barriers.Our corrections are therefore focused on getting residues over energy barriers and into the right local minimum.
As you can see in Coot, this orientation is not a terrible fit to the density - but it is a rotamer outlier and energetically unfavorable due to an eclipsed Chi angle.You can see the eclipsed conformation by looking down the CG-CB bond and noting that the CD1 atom nearly overlaps with the backbone CA.Recentering on CB may help you arrange this viewpoint.The residue also has a suggestive positive difference peak near the CD1.
To confirm the correctness of this change, use the "Undo" and "Redo" buttons to observe the change again.Look down the CG-CB bond as before. You should see that the sidechain has a favorable, staggered conformation after the change.Also note that the CD2 atom is now pointed towards the positive difference density peak - you've got the atom close enough to the correct position that refinement should be able to sort out the details.
Return to the Validation GUI window, and find the list of C-beta position outliers.Select Ile 163 from the A chain, which will center on this residue's CB atom in the Coot window.This sidechain also appears in the rotamer outliers list on this tab and on the bond-length outliers list on the Basic Geometry tab.Misfit sidechains will often have multiple diagnostic indicators of a problem, which is useful in identifying the worst offenders.Also note the large blob of positive density near to the sidechain, further evidence that it may not be in an optimal position.
Select the "Auto Fit Rotamer" and then click an atom in the Ile 163 sidechain.Alternatively, you can define a Real Space Refine Zone around this sidechain, and drag the CG2 atom of the short arm into the large difference density peak.Use the "Undo" and "Redo" to toggle back and forth across the change.Note that the CA and especially the CB atoms have moved significantly.
Correcting misfit sidechains such as these can be tricky, as many rounds of refinement within the wrong local minimum have caused distortions in the model to accommodate the misfit.It would be important to revisit this region after another refinement.In this case, you can expect to find position of this Ile further improved, as the neighboring atoms are able to recover from the strain caused by the initial outlier.
Asparagine, Glutamine, and Histidine sidechains are pseudo symmetric.They can easily be fit into electron density backwards, eliminating hydrogen bonds and introducing clashes.Hydrogen addition via Reduce (in Phenix, Coot, or MolProbity) will usually "flip" these N/Q/H residues to the positions with best steric contacts.Here, you can use Coot to force a necessary Histidine flip.
Select HIS 39 from the A chain, which will center on this residue in the Coot window.Note that the CD2 atom of His 39 and the N atom of Asp 41 are involved in a serious clash.If we flipped the His 180, then the ND1 atom would be in a position to form a hydrogen bond with the backbone NH where this clash is.
Define a Real Space Refine Zone that includes HIS 39.In the refinement menu that pops up, look under "Active Refinement" and click the "Side-chain Flip 180" button.(This flip only works on N/Q/H residues; use the Auto Fit Rotamer tool for other tasks.)
Steric clashes usually serve as emphasis on other model problems.However, there are some errors that are clearly diagnosed by clashes alone, such as N/Q/H flips and certain misplaced water molecules.There are many tools for identifying misplaced waters, and clashes are excellent for finding the most problematic ones.
Select "A 177 GLN HG3 ___ A 554 HOH O" from the list of clashes, which will center on this clash in the Coot window.Scroll the 2Fo-Fc map contour down, and note that this water has only marginal density even at a low contour.There's no justification for keeping a water in this position.
The kind of sidechain fixups you've done in KiNG or Coot can mostly be accomplished using real-space refinement in phenix.refine (which includes a rotamer correction component), up to somewhere between 2 and 2.5 (and of course NQH flips are done automatically by Reduce in either MolProbity or Phenix).At lower resolution, however, real-space refinement with rotamer correction cannot reliably discern between correct and incorrect rotamers.That's a hard job for people as well, but can often be done if you have the interactive information on clashes and H-bonds from the non-pairwise, H-aware all-atom-contact dots.
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