10.22.26 - Introduction to Structural Biology – Klemens Wild

Xray structure crystallography advantages and limitations

Advantages 

No limitation of particle size 

Atomic resolution possible 

Disadvantages 

Crystallization is heuristic 

Crystal packing is artificial 

Electron microscopy advantages and limitations

Advantages

No limitation in particle size

Physiological relevant complexes

Disadvantages

Expensive

Relatively low resolution

Nuclear magnetic resonance

nuclear resonance of nuclear spin with external magnetic field

Sample needs unpaired n x ½ nuclear spin (1H, 13C)

Very strong magnet (900 MHz)

NMR advantages and limitations

Advantages 

Sample in solution 

Molecular dynamics 

Disadvantages 

Limitation in molecular size (<30 kDa) 

Limitations in sample preparation 

electron microscopy recent developments

Advanced technologies (direct detectors, phase plates) 

Cryo cellular tomography at molecular resolution – frozen 3D cells at nm resolution 

Cryo-EM of biological complexes smaller than 200kDa 

NMR recent developments

Solid state NMR – membrane proteins and amyloids 

Advanced methods for macromolecular interaction – paramagnetic relaxation enhancement 

In vivo NMR techniques – structure determination in living bacteria 

Steps of protein structural biology 

Expression and purification – crystallization – data collection – structure determination – model building – refinement and validation 

Prerequisites for protein crystallization 

Metastable solution separating into protein-rich phase and saturated solution 

Protein available in large quantities 

Protein pure and concentrated (>5mg/mL) 

Protein in stable conformation (globular) 

Protein stable over time 

Methods of protein crystallization 

hanging drop, sitting drop, in-vivo

hanging drop protein crystallization

Well-plates filled with reservoir with precipitant 

Sealed glass slide with hanging 1:1 protein-precipitant drop 

Diffusion until supersaturation, triggering crystal formation 

sitting drop protein crystallization

Drop rests on pedestal above reservoir 

Directly beneath or around the pedestal is a larger liquid reservoir containing a higher concentration of the precipitant. 

in vivo protein crystallization circumstances

special tissues, disease, virus

crystal characteristics

Solid state 

Unit cell (three axes) 

Lattice (mathematical points) + basis (motif, molecule) 

Anisotropic (directionally dependent properties) - mechanic, optic, electric, magnetic 

Symmetrical - 230 possible symmetries (rotation, mirror, inversion) 

asymmetric unit

largest volume in unit cell without crystal symmetry 

Structure determination of a crystal means

space group determination and knowledge content of the AU 

Characteristic x rays

collision ionization of matter by fast electrons induces X-rays of element specific wavelength 

synchotron (x ray generator)

Polychromatic X-rays produced from electrons moving in a circular orbit at a relativistic speed 

four types of x ray interactions with matter

Photoelectric effect, Compton effect, Pair production, Elastic scattering (Thomson scattering)

photoelectric effect

Ionization of the atom. An outer electron is kicked out of the shell of one atom. 

compton effect

Inelastic scattering, the kinetic energy of the photon is transferred to the electron which results in a change of the wavelength. 

pair production

Formation of a positron and an electron. Only at highest energies. 

elastic scattering

mostly used for X-ray crystallography, as the kinetic 

energy and photon frequency does not change as a result of the 

scattering. Braggs law is valid and no energy deposition on the 

material.

braggs law

diffraction of the waves is given by diffraction planes of the crystal 

Diffraction image and data collection process

Each diffraction spot (reflex) corresponds to a set of lattice planes 

During data collection (exposition and rotation of a crystal in the X-ray beam) all reflexes are measured to yield a complete data set 

Crystallographic phase problem

phase information is lost during data collection, the inability to directly measure the phase angles of scattered X-rays, neutrons, or electrons during a diffraction experiment 

Model building and refinement restraints

resolution, bond lengths, bond angles (chirality, planarity), thermal motion, similarity 

R-Factor (‘reliability factor’)

Discrepancy between refined model and experiment 

types of electron scattering

elastic, unscattered, inelastic

SPA vs Tomography - different orientations in cryo EM

Single particle averaging - Multiple particles in different orientations 

Tomography - Same particle from different angles = tilt series 

When is refinement finished? 

No unexplained electron density or differences to experiment. 

No significant deviations from stereochemical targets. 

Model makes chemical and biological sense.