Using X-ray structures for bioinformatics

Robbie P. JoostenNetherlands Cancer Institute

Autumnschool 2013

Structures in bioinformatics

• Understand biology– Direct interpretation

– Data mining– Homology modeling

• Drug design• Molecular dynamics

Basic rule: Better structures → Better

results

Introduction

Right structure(s) for the job

1.Selection: find (a number of) PDB entries

2.Validation: check the quality of your selection

3.Optimisation: maximise the quality of your selection

Focus on X-ray structures

Introduction

X-ray structures have a history

1. Protein expression2. Crystallisation3. X-ray diffraction

experiment4. Model building and

refinement5. Deposition at the PDB

All these steps affect the final PDB

file

Selection

Protein expressionA ‘construct’ is made• Partial proteins– E.g. only extracellular domain of membrane protein

• Frankenstein proteins– Fusion proteins or chimeras

• Mutants are introduced – Some by accident!

• Poly-histidine tags added for purification

• Altered glycosylation state– Large sugars hamper crystallisation

History

CrystallisationThe protein stacks regularly to form a crystal• Protein still functional in the crystal

• Much solvent in the crystal (~40%)• Some residues can move– Disorder: missing loops/side chains– Alternate conformation

History

CrystallisationBeware of crystal packing• One copy of the protein can influence the next

History

CrystallisationChemicals are used for crystallisation• Buffers to stabilise the pH• Precipitants– Change solubility of the protein– Neutralise local charges– Bind water– High concentrations are used• Compounds compete with natural ligands

• Examples:– Polyethylene glycol (PEG)– Ammonium sulphate

History

CrystallisationBeware of the crystallisation conditions

History

CrystallisationBeware of the crystallisation conditions

History

X-ray diff ractionTypical experiment

History

X-ray source

Detector

X-ray diff raction• X-rays interact with electrons– Atoms with few electrons (H, Li) do not diffract well

• X-rays cause damage to the protein– Acidic groups (ASP en GLU) can be destroyed

– Disulphide bridges are broken– Hydrogens are stripped– Cooling crystals in liquid nitrogen helps• Glycerol added to the crystal!

History

X-ray diff raction• We are not using a microscope• We don’t measure everything we need

History

X-ray diffraction gives an indirect and incomplete measurement

ρ (𝑥 , 𝑦 , 𝑧 )= 1𝑉 ∑

h∑𝑘∑𝑙

𝐹h𝑘𝑙𝑒[− 2𝜋 𝑖 (h𝑥+𝑘𝑦+𝑙𝑧 )−𝛼]

MeasuredMissing: phase

Model building and refinement

Iterative process

History

Phases + calculated X-ray data

Electron density maps

Structure model

Measured X-ray diffraction

data

Initial phases

FT

FT

Model building

History

Two types of maps1. Regular electron density map (2mFo-

DFc)2. Difference map (mFo-DFc)

Model building and refinement

Fitting atoms to the ED map and trying to remove difference density peaks

HistoryModel building and refinement

• Requires skill and experience• Requires time and patience• Requires good software

Lack of any of these can be seen in the final PDB file

HistoryModel building and refinement

• Both coordinates and experimental X-ray data are deposited

• PDB standardises files and adds annotation

• Sometimes things go wrong

History

Deposition at the PDB

LINKs between alternate conformations

History

Deposition at the PDB

History

Deposition at the PDBUn-biological LINKs (in 1a1a)

LINK C ACE C 100 N PTH C 101

LINK C PTH C 101 N GLU C 102

LINK CF PTH C 101 OG SER A 188

LINK N DIP C 103 C GLU C 102

LINK C ACE D 100 N PTH D 101

LINK C PTH D 101 N GLU D 102

LINK N DIP D 103 C GLU D 102

Think of what happened to the

structure before you downloaded it

Use the experimental data• Resolution says very little about the structure

• (free) R-factor gives the overall fit of the structure to the experimental data

• For biological interpretation more detail is needed

Use the maps

Validation

X-ray specific validation

Which is the better structure of berenil bound to DNA?

Validation

X-ray specific validation

PDB id Resolution R

268d 2.0 0.160

1d63 2.0 0.183

Validation

X-ray specific validationThe real-space R-factor (RSR)• A per-residue score of how well the atoms fit the map

• Works like the R-factor (lower is better)

Maps can help distinguish the good and bad bits of a structure

Validation

X-ray specific validation

Poorly fitted side-chains

Evil peptides

ValidationThings you can find in maps

The wrong drug

ValidationThings you can find in maps

Sequence error K -> R• Accidental mutant• Also a missing sulfate

ValidationThings you can find in maps

Missing water Missing alternate conformation

ValidationThings you can find in maps

• Visualisation in Coot– http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/

• Get maps and real-space R values from the Electron Density Server– http://eds.bmc.uu.se/eds/index.html– Direct interface with Coot

• Get maps and updated models from PDB_REDO

Practical session

Validation

Checking maps

Maps show things you cannot see

otherwise

• Solved by a diverse group of scientists– People make errors & gain experience

• Since 1976– Structures are not updated

• Solved with the methods of their era– Methods improve over time

Structures in the PDB do not represent the best we can do

NOW

Optimisation

Structures in the PDB

• Take structure + experimental data• Use latest X-ray crystallography methods– Decision making: use case-specific methods

– Create new methods when needed

• Improve model quality– Fit with experimental data– Geometric quality

• Fix errorsPDB_REDO

OptimisationImprove structures in PDB

Step 1: prepare data• Clean-up structure and X-ray data• Data mining

Step 2: establish baseline• Fit with experimental data (R-factors)

• Geometric quality– Validation with WHAT_CHECK

Optimisation

PDB_REDO method

Step 3: re-refine structure (with Refmac)

• Improve fit with experimental data– Use restraints to improve geometric quality

• Improve description of protein dynamics– Concerted movement of groups of atoms (TLS)

– Anisotropic movement of individual atoms

Optimisation

PDB_REDO method

Step 4: rebuild structure • Delete nonsense waters• Flip peptide planes• Rebuild side-chains– Add missing ones– Optimise H-bonding

Step 5: validate structure • Geometry• Density map fit• Ligand interactions

Optimisation

PDB_REDO method

• www.cmbi.ru.nl/pdb_redo– > 72,000 structures (98%)– Detailed methods & reprints

• Directly in molecular graphics software– YASARA– CCP4mg– Coot (needs plugin)– PyMOL (needs plugin)

• Linked via PDBe & RCSB

Availability

PDB_REDO databank

Worse Same Better0%

25%

50%

75%

100%

8%12%

80%

Ramachandran plot

• Improved fit with the data• Better geometry

Worse Same Better0%

25%

50%

75%

100%

9%17%

74%

R-free

Worse Same Better0%

25%

50%

75%

100%

4%

22%

74%

Fine packing

Optimisation

Does it work? ( 1 2 , 0 0 0 s t r u c t u re s )

MolProbity validation ( 1 e o i )

PDB PDB_REDO

Optimisation

OptimisationElectrostatics calculations

• ‘Missing’ positive lysine atoms distort electrostatics calculations

• Adding missing atoms correctly describes C-terminus interaction with side chains

• Wrong peptide plane in peptide ligand

• Fixed by PDB_REDO• Better understanding of H-bonds in the interaction

Optimisation

Protein-ligand interaction

OptimisationProtein-protein interaction

• Packing interface with poor ionic interactions

• Rebuilt interface properly describes ionic dimerisation interactions

Optimised structures give a better view of

the biology of the protein

PDB_REDOersAmsterdam:• R Joosten• K Joosten• A Perrakis

Key contributors:Eleanor Dodson, Ian Tickle, Paul Emsley, Ethan Merritt, Elmar Krieger, Thomas Lütteke, Rachel Kramer Green, Sanchayita Sen

Nijmegen:• T te Beek• M Hekkelman• G Vriend

Cambridge:• G Murshudov• F Long