Viewing three-dimensional structures of proteins

The primary resource for obtaining the atomic coordinates of proteins/enzymes (if available) is the Protein Data Bank (http://www.rcsb.org).*

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*RCSB (established in 1971)stands for Research Collaboratory for Structural BioinformaticsThe Protein Data Bank is managed by the Worldwide Protein Data Bank

Viewing three-dimensional structures of proteins

As of September 13, 2020 there are 168,600 macromolecular structures deposited in the data bank.

The structures include those of proteins, nucleic acids, protein-protein adducts, protein-nucleic acid adducts, adducts with small molecules (inhibitors, drugs, metals, etc.).

The data is typically obtained by X-ray crystallography (majority of cases), by NMR spectroscopy (suitable for smaller macromolecules), and more recently by cryo-electron microscopy.

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ExperimentalMethod Proteins Nucleic Acids Protein/Nucleic Acid

complexes Other Total

X‐ray diffraction  132304 2082 7024 7973 149383

NMR 11458 1300 265 92 13115

Electron microscopy 3957 47 1362 455 5821

Hybrid 160 5 3 6 174

Neutron 67 2 0 0 69

Other 32 1 0 4 37

Total: 147978 3437 8654 8530 168599

Breakdown as of September 13, 2020 (https://www.rcsb.org/stats/summary)

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PDB: 1IAGFIRST STRUCTURE OF A SNAKE VENOM METALLOPROTEINASE

Zn2+

SO42-

H2O

-helix

strand

loop

Zinc-bound H2O

Adamalysin II (24 kDa zinc endopeptidase)

Generated from 1IAG using Discovery Studio 4.1 Visualizer (formerly Accelrys, now Biovia)

http://www.3dsbiovia.com/products/collaborative-science/biovia-discovery-studio/visualization-download.php

Ribbon representation (with active site highlighted)

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C wire representation(shows protein

backbone)

Line representation

(shows all atoms except hydrogen)

Adamalysin II – Alternative representations

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Ball-and-stick representation

(shows all atoms except H atoms)

Spacefillrepresentation(shows atoms as VdW spheres)

Adamalysin II – Alternative representations

What are the benefits of each representation ?

Ca(II)

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A note on conventional colour coding:

Grey – carbon atomsRed – oxygen atomsBlue – nitrogen atomsYellow – sulfur atoms

Other colours can be chosen for other elements (e.g., metal ions)

For surface (atom charge) representations:

Grey – neutralRed – negatively chargedBlue – positively charged

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Stereoview (cross-eyed) (provides a 3-dim view of the structure)

For right eye For left eye

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Free software suitable for viewing and manipulating pdb files:

PyMOLRasMolChimeraVMDDiscovery Studio Visualizeretc.

(see https://www.rcsb.org/pages/thirdparty/molecular_graphics for a more comprehensive list)

Still in development: Free software for the visualization of structures in virtual reality (VR)* – allows for an immersive experience of 3d environments

* Nanome introduced a free version (with limited capabilities), but requires more expensive gear (not just Google cardboard)

Nanome: main protease of Covid‐19(https://www.youtube.com/watch?v=KOchIPVUE_E&list=PLjQh_Yi8CoBzPNjCAU3aIvGwPrOU_‐BrC&index=2&ab_channel=Nanome)

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Single crystal diffraction pattern of lysozyme

X-ray crystallography

Used to determine the 3-dim structure of crystals (incl. minerals, proteins, etc.)

sin Θ = xy / d

constructive interference:

d = lattice plane spacing

typically:(X-ray) = 1.54 Å (Copper – K)

X-rays are scattered (diffracted) by the electrons of atoms. The heavier the atom (the more electrons it has), the higher the amplitude of the scattered wave.

10From: De Mattei, R.C. et al. (2001) J. Cryst. Growth 232, 511-519

crystal grown in space(better crystal, better resolution)

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Electron density contour map and model of the metal-binding site in a Zn finger protein

X-ray crystallography

Processing of the diffraction pattern (each spot is analyzed in terms of position and intensity) yields electron density maps.

Processing

Density maps are then used to model atoms, amino acids, back-bone, side-chains, cofactors, small molecule inhibitors, etc.

Resolution of a crystal structure (depends mainly on the quality of the crystal):1.2 Å Excellent backbone and most sidechains are very clear (some H

atoms may be resolved)2.5 Å Good backbone and many sidechains are clear3.5 Å OK backbone and bulky sidechains are mostly clear5.0 Å Poor backbone mostly clear but sidechains are not clear

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1.2 Å Excellent backbone and most sidechains are very clear (some H atoms may be resolved)

2.5 Å Good backbone and many sidechains are clear3.5 Å OK backbone and bulky sidechains are mostly clear5.0 Å Poor backbone mostly clear but sidechains are not clear

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NMR spectroscopy

For solving structures of smaller (< 40 kDa) proteins or macromolecules

Often used when the protein is difficult to crystallize, or to obtain the solution structure

Basis of NMR spectroscopy

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NMR spectroscopy

Nuclear Overhauser effect (NOE). With the help of the NOE, pairs of protons that are in close proximity can be identified.

Inducing a transient magnetization in a sample by applying a radio-frequency pulse leads to a change in the spin of one nucleus. Examining the effect on spins of nuclei in the vicinity forms the basis of NOESY.

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Detection of short proton–proton distances by NOESY. The off-diagonal peaks corresponds to a short proton–protonseparation.

Calculation of structure based on NOESY experiments: (A) NOESY shows the protons connected by the red lines to be close to each other. (B) A 3-dim structure is calculated based on the constraint that the proton pairs identified in (A) are close together.

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A set of 25 conformer structures for a 28 AA domain from a zinc-finger-DNA-binding protein. Magenta line = average of the protein backbone

pdb: 2M9QNMR structure of an inhibitor-bound dengue NS3 protease

NMR structures look somewhat different from X-ray structures in that a family of structures is obtained.

The differences between individual structures are due to: imperfections in the experimental data dynamic nature of proteins in solution