practical crystallography from protein to structure

how are protein crystals held together

weak intermolecular forces:

van der Waals

polar

H-bonds

hydrophobic pi-stacking

electrostatic

what is in a crystallisation solution that is mixed with concentrated protein

salts

pH buffer

precipitant to compete with protein for space in solution

additives to help protein function

crystallisation experiment

mix equal volume of concentration protein solution and crystallisation solution in a hanging drop vapour diffusion experiment

concentration of both is immediately halved

drop is sealed so water vapour is free to diffuse to a reservoir containing only crystallisation solution

as this happens, both protein and precipitant increase in conc with time and drop is saturated with protein

leads to spontaneous nucleation in the metastable zone of crystallisation phase diagram

as crystals grow bigger in experiment

protein conc drops

vapor diffusion continues until an equilibrium is reached

final coordinate on phase diagram at equilibri8um determines outcome of experiment

unsaturated zone

clear protein solution

metastable zone

will eventually separate into proteins (maybe crystals) and saturated solution

precipitation zone

unstable, will spontaneously decompose into precipitate and protein solution

steps for performing diffraction experiment

cryoprotectant (glycerol) added into drop

crystals are fished out of drop using nylon loops and cryo-cooled in liquid nitrogen

crystals are shot with x-rays, generated by a rotating copper anode or a synchotron

setup for shooting x-rays at crystals

crystals mounted on goniometer

cyrostream blows 100k N2 gas over crystal

detector records diffraction spots

beamstop prevents incident beam damaging the detector

crystals are rotated in the beam to give all bragg planes a chance to diffract

processing diffraction data

indexing - find spots and assign to bragg planes, spacing of spot gives dimension of unit cell

integration - add all pixel counts in each spot to give intensities (amplitudes of diffraction vectors)

diffraction pattern has same point-group symmetry as crystal - symmetry related planes give reflections with equal intensities - can average together to determine rotational symmetry and space group

solve phase problem - use molecular replacement or experimental phasing (using heavy atoms)