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Assessing structure quality in the PDB archive

Presenters: Matthew ConroyHost: Adam Carter

BioExcel Webinar Series

8 February, 2017

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Today’s Presenter

Matthew Conroy is a Scientific Curator at EMBL-EBI working with the Protein Data Bank in Europe. Before joining PDBe, he was solving structures of proteins by X-ray crystallography, NMR and electron microscopy.

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Protein Data Bank in Europe

PDBe.org

Assessing structure quality in the PDB archive

Matthew Conroy

What is the Protein Data Bank (PDB)?

PDBe.org

An archive of experimentally determined 3-dimensional structures of biological macromolecules

Protein, nucleic acids, sugars

wwPDB.org

‘The PDB’FTP Archive

wwPDB.org

‘The PDB’FTP Archive

Value added data

Value added data

Value added data

Value added data

From the small… …to the large

Models are interpretations of experimental data

PDBe.org

Copper scavenger <1kDa

Zika virus 4 MDa

What experimental data are available?

PDBe.org

10% solution NMR SpectroscopyData: Restraints (mandatory since 2008)

Chemical shifts (mand. since 2010)

88% X-ray crystallographyData: Structure factors (mandatory since 2008)

1% Electron microscopyData: Map in EMDB (mandatory since 2016)

Like in all science:

Some data are better than other data

Some models (interpretations of that data) are better than other models

For whatever you need to do, you need to:Find the best modelBe aware of any potential limitations

Different techniques have different strengths

• “Structures are not absolute truths – they are models that fit the experimental data and therefore have uncertainty and subjectivity associated with them.”

Whaddaya Know: A Guide to Uncertainty and Subjectivity in Structural Biology

Trends in Biochemical SciencesMacKay et al 2017

DOI: 10.1016/j.tibs.2016.11.002

Poll

• What do you use PDB data for primarily?

• Template for homology model

• Protein-Protein complex prediction

• Small molecule (drug) docking studies

• Something else

All structures in the PDB are models explaining data

PDB archive does not reject structures based on qualityBut we do give an indication of that quality

• Essentially, all models are wrong, but some are useful.

• George E.P. Box

Validating PDB Data

What is the wwPDB doing to indicate structure quality?

Validation Task Forces

Method-specific Validation Task Forces

Advise us how best to validate:

• the models

• the experimental data

• fit of model to data

Most entries in the PDB archive come withvalidation reportsPDF documents downloadable from wwPDB sitesAlso XML format for machine reading

Model Quality Data Quality* Fit of model to data*

X-ray ✓ ✓ ✓

NMR ✓ ✓ ✓

EM ✓ Not yet, but on EBI’s EMDB pages

Not yet, but on EBI’s EMDB pages

* If data were deposited

On wwPDB sites(recalculated annually)

Available at deposition(for authors and referees)

Standalone servervalidate.wwpdb.org

At PDBe, they’re available from search results

PDBe.org

And also from each entry page

PDBe.org

Start with a summary and gets more detailed

1. Overall quality at a glance

2. Residue-property plots

Highlight outliers

3. Detailed analysis of any potential issues

Start with a summary

• Overall quality at a glance

How does this structure compare to others in the PDB archive?

Several different metrics considered

Poor ranking doesn’t necessarily mean a structure is ‘wrong’

It may mean it’s not as ‘right’ as others

Justified reasons for outliers eg strainAre they supported by data?

PDBe.org

We use these to rank results at PDBe

Quality

PDBe.org

Overall quality at a glanceaka ‘Summary Sliders’

Atoms bumping into each other

Geometric quality assessed using MolprobityTo compare a structure to others in the PDB

X-ray, NMR and EM are all judged the same way

Surprising bond angles Compared to all PDB

and to similar structures

Of course, these only tell you the model is chemically sensible

Good geometry doesn’t mean it’s right!

Overall quality at a glanceaka ‘Summary Sliders’

Extra metrics for X-ray structuresHow well does the model back-predict the data?Lower values are better

Residues not in electron density

The real-space R-value (RSR) measures fit between a residue and the data. The RSR Z-score (RSRZ) is a normalisation of this specific to the residue type and a resolution bin. Outliers have RSRZ >2

A look at X-ray data

From the diffraction pattern, a map can be calculated.

This indicates the location of electrons (therefore atoms) in the crystalHence- ‘electron density map’

Model is built into this map in an iterative process

Resolution indicates precision with which atoms can be placed

3.7Å

2.4Å

1.5Å

0.8Å

In low resolution data, models might simply indicate location/orientation of proteins

37Å EM map of F1Fo ATPase to show role in shaping mitochondrial cristae

PDB entry 4b2q

Colour coded according to the number ofgeometric quality criteria outliersie model quality

Green = 0Yellow = 1Orange = 2Red = 3 or more.Grey = unmodelled residues

Outliers in these metrics:• bond length & angle • Chirality/planarity • too-close contacts • Ramachandran• sidechain torsion angle• RNA sugar pucker

Residue-property plots – per polymer chain

The red dot! For X-ray structuresA red dot above a residue indicates a poor fit to the electron density

(ie RSRZ outlier)

Visualising validation at PDBe

PDBe.org

PDBe.org

Like

lihoo

d of

di

sord

er

Disordered in model ensembleOrdered in model ensemble

NMR data quality and fit to data• This is at the moment basic

1. Completeness of resonance assignments% of atoms for which chemical shift is measured

2. Statistically unusual shifts

3. Random Coil Index• Do chemical shift and protein conformation agree?

EM data quality and fit to data

PDBe.org/EMD-8116

Not yet in report, but available at EMDBpdbe.org/emdb

Geometry and fit to data go hand-in-hand

Asp 62 has some outlier bond lengths

But these are not justified by the electron density

Val 48 in entry 3kse is a Ramachandran outlier

But the strained conformation is supported by data

An aside on Assemblies• Only the smallest part of a

crystal structure is deposited to the PDB archive

• The whole can be generated by applying symmetry to this

• The part you’re interested in could be:

The file as it is:

The file and symmetry

Only part of the file

HIV protease. Entry 2az9

Viewing assemblies at PDBe with LiteMol

PDBe.org

Validation for small molecules

• There are thousands of amino acids and nucleic acid bases in the PDB

• But a small molecule could be unique

• So how can we tell what it should look like?

Summary of issues: X marks an outlier!

Is it the correct handedness?

Does it hit anything?

Is it chemically sensible?

Does it fit the data?

Validation for small molecules

PDBe.org

1. Geometry

Compares bond lengths and angleswith chemically similar fragments

Mogul - A knowledge-based library of molecular geometry derived from the Cambridge Structural Database (CSD)

Validation for small molecules

PDBe.org

2. Fit to data

RSR: Measure of how well a residue fits its local density >0.5 means it is worth a second look

LLDF- ‘local ligand density fit’Z-score of ligand RSR relative to nearby polymeric residues > 2 is flagged as unusual

LLDF: “Is the ligand data quality comparable to that of the binding site?”

Difference density- the ‘red and green bits’!Along with the electron density maps we’ve already seen, comes a second mapIndicating:

Areas where the model has too many atoms for the data

Data suggest too few atoms here

Areas where the model has too few atoms for the data

Is there a hint of a ligand bound to the Haem of this cytochrome P450?

Difference density can indicate modelling errors

Large green ‘blob’- there might be something else here

Difference density can indicate modelling errors

Aspirin in red density- presence unsupported by data(also not interacting with protein much!)

Viewing ligands at PDBe- each has its own page

PDBe.org

A summary of validation

PDBe.org

It’s not black and white! Structures can’t be assigned ‘good’ or ‘bad’ easily

Look at the validation reportHow many outliers? Are they justified? Are they talked about in the paper?Does the model fit the data?

Does the structure adequately explain what is known biologically?

A summary of validation

PDBe.org

wwPDB validation reports are extensive and a good aid to identifying structure quality

Search results at PDBe rank by quality Take into account validation metrics and resolution

PDBe pages allow validation to be viewed in 3DGeometry + data for EM and X-ray

Also a PyMOL plugin to show geometry

Available at pymolwiki.org

PDBe.orgproteindatabank@PDBeurope

pdbhelp@ebi.ac.uk

Thanks!

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Next Webinar15th February, 201715:00 GMT / 16:00 CET

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Gydo van Zundertwith Mikael Trellet and JörgSchaarschmidt

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