Structures and Structure Descriptions Chapter 8 Protein Bioinformatics.
Using X-ray structures for bioinformatics
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Using X-ray structures for bioinformatics
Robbie P. JoostenNetherlands Cancer Institute
Autumnschool 2013
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Structures in bioinformatics
• Understand biology– Direct interpretation
– Data mining– Homology modeling
• Drug design• Molecular dynamics
Basic rule: Better structures → Better
results
Introduction
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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
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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
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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
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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
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CrystallisationBeware of crystal packing• One copy of the protein can influence the next
History
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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
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CrystallisationBeware of the crystallisation conditions
History
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CrystallisationBeware of the crystallisation conditions
History
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X-ray diffractionTypical experiment
History
X-ray source
Detector
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X-ray diffraction• 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
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X-ray diffraction• 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∑𝑙𝐹h𝑘𝑙𝑒[− 2𝜋 𝑖 (h𝑥+𝑘𝑦+𝑙𝑧 )−𝛼]
MeasuredMissing: phase
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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
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History
Two types of maps1. Regular electron density map (2mFo-
DFc)2. Difference map (mFo-DFc)
Model building and refinement
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Fitting atoms to the ED map and trying to remove difference density peaks
HistoryModel building and refinement
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• 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
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• Both coordinates and experimental X-ray data are deposited
• PDB standardises files and adds annotation
• Sometimes things go wrong
History
Deposition at the PDB
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LINKs between alternate conformations
History
Deposition at the PDB
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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
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Think of what happened to the
structure before you downloaded it
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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
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Which is the better structure of berenil bound to DNA?
Validation
X-ray specific validation
PDB id Resolution R268d 2.0 0.1601d63 2.0 0.183
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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)
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Maps can help distinguish the good and bad bits of a structure
Validation
X-ray specific validation
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Poorly fitted side-chains
Evil peptides
ValidationThings you can find in maps
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The wrong drug
ValidationThings you can find in maps
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Sequence error K -> R• Accidental mutant• Also a missing sulfate
ValidationThings you can find in maps
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Missing water Missing alternate conformation
ValidationThings you can find in maps
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• 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
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Maps show things you cannot see
otherwise
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• 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
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• 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
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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
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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
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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
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• 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
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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 )
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MolProbity validation ( 1 e o i )
PDB PDB_REDO
Optimisation
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OptimisationElectrostatics calculations
• ‘Missing’ positive lysine atoms distort electrostatics calculations
• Adding missing atoms correctly describes C-terminus interaction with side chains
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• Wrong peptide plane in peptide ligand
• Fixed by PDB_REDO• Better understanding of H-bonds in the interaction
Optimisation
Protein-ligand interaction
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OptimisationProtein-protein interaction
• Packing interface with poor ionic interactions
• Rebuilt interface properly describes ionic dimerisation interactions
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Optimised structures give a better view of
the biology of the protein
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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