Structure data
Structure data
Objectives
- Understand that "sequence" and "structure" are abstractions of biopolymers.
- Understand that "structure" is an idealized concept, applied to an ensemble of dynamic molecules.
- Be aware of principal methods of experimental structure determination and some of their limitations regarding interpretation of data and resulting accuracy.
- Understand that structures may have considerable local and global uncertainties.
- Know that structure abstractions can be stored, retrieved and visualized and become familiar with the principal databases and information sources for that purpose.
- Be familiar with the contents of a PDB formatted file.
Links
- Bernhard Rupp's introduction to crystal structure
- The phase problem (of crystallography)
- Nuclear Overhauser Effect
- Whatcheck
- Procheck
- The PDB
- The PQS service
- The Nucleic Acid Structure Database
- PDBsum
- the HIC-Up database of hetero compounds in PDB files
- th JME molecular editor
- CORINA 3D-structure generator
- VMD
Slides
The "structure" abstraction
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The letter Y represents the properties of the molecule "tyrosine" in a highly compressed way.
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Which amino acid we regard as being similar to tyrosine depends on which property we are considering.
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Structure contextualizes sequence. The sequence provides a description of the molecule, but the role of the individual amino acids can only be understood in the context of their environment.
Experimental determination of structure
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See: Bernhard Rupp's introduction to crystal structure.
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The phase problem (of crystallography)
The inability to measure the phases of diffracted photons prevents the reconstruction of the diffracting objects from on set of experimental measurements alone. Additional information must be sought, based on the fact that photons that are in phase enhance the measured intensities, whereas photons that are phase-shifted by 180° cancel each other's intensities. Thus measuring intensity changes caused by additional diffraction centres that are placed into the crystall lattice, allows us to infer relative phases. If several relative phases are known, we can triangulate their absolute values. Experimental error makes this a difficult problem, but under favourable circumstances the electron density map will be interpretable; a structural model can then be built and refined.
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NMR spectroscopy is an important alternative to x-ray crystallogaiphic determination of protein structure.
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Which precessing proton resonates with a particular frequency can be determined ("peak assignment"). When the proton is excited at this frequency, its spin-polarization can be transferred through space with the so-called Nuclear Overhauser Effect. This effect is highly sensitive to spatial separation, therefore it can generate a list of distance constraints between specific protons.
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Distance constraints are translated into "pseudo energies" that are used in molecular dynamics simulations to generate structural models. The simulation starts from a random conformation and then runs until the energy of the model is minimzed. Violations of stereochemistry (bond-lengths, angles and steric clashes) are minimized together with violations of experimental distance constraints. This typically generates an ensemble of conformational models which is then averaged and further refined to finally arrive at a final consensus model.
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Structure database contents
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The PDB
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Additional complications arise from "insertion codes". These are letters that allow the insertion of residues for a common numbering scheme for families of homologous sequences. In principle this is a good idea, since this makes comparison of residues much easier. But strings such as "23A" can no longer be tretaed as "sequence numbers - thet are sequence labels and using them correctly can be a challenge.
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Potential pitfalls:
- Record type: changes not consistently applied for modifications
- Atom number: rarely used and a nuisance to update when changing.
- Atom name: careful about columns
- Amino acid type: careful about e.g. selenocysteine. Some very old files use TRY for TRP
- Chain: may be blank (" ") in older files. Recently was changed to ("A") even in files that contain only a single chain.
- Alternate location: only sometimes given in very high resolution structures.
- Sequence number
- X,Y, and Z are given in Å (10<sup-10 m = 0.1 nm) values in a cartesian (i.e. orthogonal) coordinate system; but origin and orientation is arbitrary!
- Occupancy can describe: special locations, partially bound ligands, unobserved fragments of structure
- B-values, (also called temperature factors) are a measure of the volume of space around into which a
Read the Coordinate section of the PDB format specification (V 2.3)
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See the HIC-Up database of hetero compounds in PDB files.
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Creating small molecule structures from scratch with th JME molecular editor and the CORINA 3D-structure generator.
ACD chemsketch is a windows-only alternative.
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Asymmetric units may contain only a single monomer of a homooligomer.
In the example above, only one chain of the tet-repressor dimer is seen bound to only one strand of B-DNA. The second chain and strand can be generated through a symmetry operation (180° rotation and translation), thus it contains the same coordinate information and does not need to be separately stored. However, in order to study the functional molecule, the redundant coordinates have to be combined to a homodimer. The term biological unit describes a coordinate set that (presumably) depicts a homooligomer in its functional state. All molecules in the crystal lattice can be generated from the crystallographic symmetry operations specified in a PDB file, for the space group of the crystal. But it may not be obvious which of the symmetry replicates might actually be involved in a physiological interaction and which ones have only been induced by the crystallization process. In the tet-repressor example, the crystallographic space group has eight symmetry related monomers in the unit cell of the crystal lattice.
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The PQS service for Probable Quarternary Structures at the EBI.
Many biological unit structures are also made available directly from the structure summary page of the PDB Website.
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The Nucleic Acid Structure Database
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PDBsum is a secondary database thst stores analysis and interpretation information for PDB coordinate sets.
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Various options for visualization give different levels of abstraction, from space-filling model, to line drawings that emphasize chemical connectivity, to tube or cartoon models that trace the overall folding topology of a protein.
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Various tools exist for different visualization tasks. ORTEP was one of the earliest programs and plots thermal ellipsoids with three degrees of freedom. Most protein structures do not have this data available but many small molecule structures do. There is a host of 3D, interactive molecular visualization programs available, in this course we use VMD. Examples of programs that draw moleculer scenes for publications include Molscript, or the generic ray-tracing program POVray. VMD can generate povray input files, such as the one from which I generated this internally illuminated view of Green Fluorescent Protein (GFP).
