Methods

hundreds of million know protein sequences - numbers grows

3D structure information is encoded in the sequence

models for molecular replacement

protein/peptide complexes

structures of oligomeres

discovery of new protein fold classes

> guiding experimental strucural biology

target structures for rational drug design

no crystals needed

buffer choice highly flexible

can deal with unstructured regions in proteins

dynamic information on multiple time scales

great for testing ligand binding and biomolecular interactions

upper size limit for structure determination (30 kDa)

large amounts of sample required

Isotope-labelling required

long data aquisition times

labor-intensive data analysis

no crystals needed

no upper size limit

only very small amount of sample needed

buffer conditions adaptable

if complexes are at multiple functional states at the same time (e.g. different conformations etc.)

> at the sorting step you can also start by state (not only by direction) that are also functionally relevant

lower size limits (>50kDa preffered, bigger = easier)

not yet at true atomic resolution

data analysis not yet standardized, complex software packages

high energy electrons collide with a metal target

eject electrons from the inner electronic orbitals of the metal atoms

electrons from the outer orbitals fill the empty positions, emit X-rays

X-ray sources:

• synchroton radiation in particle accelerations

  • very intense X-ray emission

  • small crystals

  • short measurement times

  • variable wavelengths

true atomic resolution possible

no size limitations

mathematically rigorous protocols for converting measured data into electron density

highly standardized mature software packages

crystals needed extensive screening efforts

buffer conditions not free selectable

difficulties with proteins that contain large unstructured domains

large amounts of sample needed

3D structures of proteins, RNA, DNA and their complexes

> large macromolecules assemblies (Ribosome, proteasome and RNA-polymerase)

no size limitation

high quality crystals

wavelength of 0,1 - 100 Å

high water content - native like environment

crystallization normally doesnt change the protein structure

many proteins aee enzymatically active in the crystal or still able to bind ligands

for many proteins different crystals forms exist

> rearrangement of the individual protein molecules with repect to each other differs as well as the intramolecular contacts

thermodynamicalls stable

Method: A hot, saturated solution is cooled down slowly, or a non-polar organic solvent is added to an aqueous solution

Problem: these conditions are too drastic for proteins, as they would cause the protein do denature

depends on precipitant concentration

temperature

pH

salt concentration

in 3 dimensions highly ordered, repetitive arrangement of the biological macromolecule

membrane proteins: in some cases - 2D crystals, strucutre determination possible with specific techniques (electron diffraction)

high water content compared to salt crystals (50-70%)

much smaller (10 µm to 300 µm)

microfocus synchroton beamlines: - 5 µM in the smallest dimension

stabilized by weak non-covalent intramolecular interactions (hydrogen bonds)

high water content

dried protein looesed structural integrity

alternative: quick freezing of crystals in liquid nitrogen or ethane in the presence of “cryoprotectants”

ordered water: bin to the protein in the same position