Introduction to Structural Bioinformatics

1
Introduction to Structural Bioinformatics

• Bruce Byrne, PhD
• Fundamentals of Bioinformatics
• Spring, 2007

2
What we will be doing

• The Power Point Presentation
• Whats important about structural bioinformatics?
• There are themes in common to the rest of
  bioinformatics.
• How do we get structural information for
  proteins?
• Laboratory techniques
• Data search and retrieval
• What do the data look like?
• How can we visualize the data?

3
Structural Bioinformatics

• Why might it be useful to understand both the
  sequence and structure of a protein?
• Better understanding of functional interactions
• Protein/ligand binding
• Catalysis
• Insight into aberrant biological phenomena
  (disease)
• Cancer, diabetes, etc
• Develop drugs
• Control disease by interfering with key pathways
  at molecular level
• Influence disease course with metabolic
  inhibitors/activators
• Manipulate gene and protein
• Gene therapy
• Biopharmaceuticals

4
Pattern Recognition is Fundamental to
Bioinformatics

• Texts and Sequences
• Text exact matches to words or strings
• Sequence similarity
• Expression Data DNA Microarray
• Experimental data represented by color and
  intensity. How do patterns compare from on
  condition to another?
• More, later, in Gene Expression unit.
• Molecular Structures
• How can a characteristic set of amino acids,
  arranged in 3-D, account for ligand or receptor
  binding?

Text
Sequence
Expression
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What Happens with Advanced Structural
Bioinformatics?

• Now
• Manipulating sequence or altering structure
• Modeling a similar sequences with unknown
  structure to a known structure
• Docking a macromolecule and ligand
• Future
• Predicting structure based solely on sequence
• ab initio methods

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Ab initio methods

• Build a peptide structure with just sequence
  information.
• Can have more than one low energy model.
• Iterative process and convergence is a must
• Feasible for short peptide sequences (best model
  was 112aa by Robetta server (David Baker, Nature,
  2007)
• Useful to build loop regions, missing protein
  segments in low resolution structures.
• Complimentary to NMR and X-ray crystallography.

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Basics Data for Modeling Structure

• Solution NMR Nuclear Magnetic Resonance
• Does not require crystallization
• Works on molecules in aqueous solution
• Lower resolution
• X-Ray Crystallography
• 80 of PDB entries
• High resolution
• Large quantities of crystallized protein
• Validity for some biochemical microenvironments?
• Highly hydrated crystals
• Good empirical agreement between solution NMR and
  crystallography

8
Imaging - General
Image in and out of focus

• Ability to create an image related to wavelength
  of energy source
• Light
• X-rays
• Neutron beams
• Electrons
• Lenses focus images using a variety of energy
  sources
• Glass light
• Magnets electrons
• Diffraction creates patterns
• Regular, interpretable patterns resulting from
  the interference of waves

Waves on the surface of water diffracted moving
through a small hole
Given knowledge of the pattern on the left
and the properties of the hole, the waves on the
right can be modeled
Images Wikipedia
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X-Ray Crystallography

• Not just an imaging technique
• Data gathering
• Substantial interpretation
• How does it work?
• Wavelength (Ångström range, 10-8 cm ) will cause
  scatter by electron cloud of similar sized atom
• Generally yields a unique model
• Cannot resolve the positions of hydrogen atoms
  unless by modeling or resolution beyond about 1.2
  Å
• Terminal side-chain atoms uncertain for Asp, Gln
  and Thr requires inferred identity

10
Solution NMR

• Analyzes proteins in solution
• Especially useful for smaller proteins, lt 30 kD
• Very important because
• some proteins resist crystallization
• Yields the positions of some hydrogen atoms
• Solution NMR often yields multiple models, in
  comparison with crystallography
• Especially useful in the analysis of large
  complexes

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The Crystal

• Crystal must be
• Single
• A few tenths of a mm in each direction
• Protein crystals are
• Fragile and sensitive
• Bound by weak hydrogen bonds, salt bridges and
  hydrophobic interactions
• Contain 50 solvent in channels between stacked
  molecules
• Jelly-like nature permits soaking crystals in
  metal solutions or enzyme inhibitors
• Expensive

12
Obtaining a Crystal

• High concentration, purified protein (2-50 mg/ml
  )
• Add agents to reduce solubility, without
  precipitation
• Evaporate agent from reservoir into hanging drop
  with protein
• Experiment trial and error

13
The Experimental Set-up

• Rotating anode X-ray generator
• Monochromator or focusing mirrors yield single
  wavelength
• Crystal can be repositioned using goinometer
• Photo-plate or electronic recording of diffracted
  pattern

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Interpretation Theory Bragg's Law

• n? 2d sinT (1)
• Derived by Sir W.H. Bragg and his son Sir W.L.
  Bragg in 1913
• Explains why crystals reflect X-ray beams at
  certain angles of incidence (theta, q).
• Direct evidence for the periodic atomic structure
  of crystals postulated for several centuries.
• The Braggs were awarded the Nobel Prize in
  physics in 1915.

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Interpretation Practice

• Obtain diffraction pattern
• Position and intensity apparent in image
• Phases of the waves which formed each spot must
  also be determined
• irradiate two or more derivatives of the same
  crystal which differ only in the presence of
  heavy metal ions
• Use multiple wavelengths
• Position, density and phase constitute a
  structure factor.
• PDB structure factor data files permit creation
  of a complete electron density map

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Structural Bioinformatics Repositories

• PDB Protein Database
• Curated collection
• Prime source for data
• NCBI
• Derived database, a subset of PDB
• One useful structural viewer
• Integrated with other NCBI databases

17
Finding The PDB File

• PDB http//www.rcsb.org/pdb/home/home.do
• Search for a PDB ID 1zaa

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Looking at the PDB Data File

• Note Display File options
• Select PDB File
• Note file structure and similarities to GenBank
• HEADER
• TITLE
• COMPND
• SOURCE
• KEYWDS
• EXPDTA etc
• REMARK
• DBREF
• SEQRES
• etc
• Far down in the file, you will note the position
  of each heavy atom of each residue of the protein
  using XYZ coordinates
• How do these data compare to what you have
  learned about X-ray crystallography?

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Finding Data

• What are Zinc Fingers?
• Navigating structures at NCBI

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Zinc Fingers (1)

• Well-understood structure with important
  biological function
• Independently folded domain of many proteins
• Requires 1 or more Zinc ions
• A series of Zinc Fingers recognizes specific DNA
  sequences
• Matches regulatory proteins like transcription
  factors

21
Zinc Fingers (2)

• Very common DNA-binding motif
• Characterized by two anti-parallel beta strands
  followed by an alpha helix
• Stabilized by Zn ion interacting with conserved
  histidine (H) and cysteine (C) residues.

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Search Using Alternative Strategies

• Use Entrez
• Search term zinc finger
• Cick structure
• 600 hits

• TaxBrowser
• Select Mus musculus
• Click structures
• Search for zinc finger
• Search for zinc finger

23
Examine the Tabs

• Tabs sort your result depending on the
• Search strategy
• Characteristics of the database
• Structure searches
• NMR
• X-ray

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MMDB Summary Page (1)

• Note layout and features
• Reference often but not always a publication
• Search resulted from match of key words, but not
  a well controlled vocabulary
• MMDB and PDB index numbers

25
MMDB Summary Page (2)

• Chains (Proteins and Nucleotides, in this
  example)
• 3d Domains
• Domain Families

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ExerciseCn3D The NCBI Viewer

• Having reviewed how structural data are obtained
  in the laboratory and catalogued at NCBI this
  Units Exercise will give you expertise in using
  the NCBI structural viewer, Cn3D.
• Download the assignement from WebCT

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Next Week Beyond

• On-line
• Find structures at NCBI using Entrez tools
• Use Cn3D Viewer to visualize structures
• Use similarity searching tools to find similar
  structures
• Review remaining schedule of classes.