protein crystals

cellulasesubtilisin

The color you see is“birefringence”, the wavelength-dependent rotation of polarizedlight.

~1mm

Crystallization robotHigh-throughput crystallography labsuse pipeting robots to explorethousnds of “conditions”. Eachcondition is a formulation of thecrystal drop and the reservoirsolution.

Conditions can have different:

•protein concentration

•pH

•precipitant, precipitant concentration

•detergents

•organic co-solvents

•metal ions

•ligands

•concentration gradient

Protein crystal growth

blue line = saturation of protein

red line = supersaturation limit

Crystal growth occurs betweenthese two limits. Above thesupersaturation limit, proteinsform only disordered precipitate.

prot

ein

conc

entra

tion

precipitant concentration

Arrows indicatedifferent diffusionexperients.

A,B,D,F,G. Vapordiffusion.

E. Bulk

C. Microdialysis

L=liquidS=solidm=metastable state(supersaturated)

vapor diffusion setup

Volatiles (i.e. water) evaporate from one surface andcondence on the other.

Drop has higher water concentration than reservoir, sodrop slowly evaporates.

a Linbro plate

Other ways to supersaturateslowly

Sitting drops

Microdialysis

Gel filtration

precipitantsA precipitant (r) causes proteins (p) to stick to each other bycompeting for solvent.

pp

r r r

pp

r r r

r = EtOH, (NH4)2SO4, methylpentanediol, polyethylene glycol, etc

http://www.ccp14.ac.uk/ccp/web-mirrors/llnlrupp/crystal_lab/hampton_screen.htm

50 of the most successful crystallization conditions:

Crystallization theoryNucleation takes higher concentration than crystalgrowth.

R RR R

R

R

R RR

R RRR

slow slow fast

RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR

R

RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR

RRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR

fastnot so slow

After nucleation, the large size of a face makes the weakbond more likely.

Periodic Bond Chain theory

Bonds A,B are stronger than P,Q. Dimensions ofcrystal at equilibrium are proportional.

More on Periodic Bond Chain theory: http://www.che.utoledo.edu/nadarajah/webpages/PBC.htm

Periodic Bond Chain theory

Growth is unfavorable directions increases as the crystalgrows.

Weak bonds in Zfavor growth in XY,forming “plate” xtal.

Growing cross-section in XYfavors growth in Z.

Ratio of crosssections isinverse to ratio ofbond strength.

diffusion depletionCrystal growth depletes the surrounding solution ofprotein, while concentrating impurities.Local depletion...

...prevents nucleations close to a growing crystal

...slows and eventually stops crystal growth

...concentrates impurities on the surface of the crystal

...causes convection currents.

Cobalt impurities in SiO2(amethyst) are concentratedin the part of the crystal that

formed last (the tip).

Better crystals in micro gravity?

More at: http://science.nasa.gov/ssl/msad/pcg/

Higher concentration of protein = higher density

Differences in densty cause convection currents, whichmight cause crystal defects. Microgravity eliminatesconvection currents.

mounting crystalsThin-walled glass capillaries(<1mm in diameter) are filledwith “mother liquor”(the fluidin which the crystal was grown)and a crystal is carefullydropped in. The mother liquoris removed using filter papercut to fine strips. The crystalsticks to the glass,immobilized.

The xtal remains in vapordiffusion contact with themother liquor. If not it willdryout and crack.

Protein crystalsare extremelyfragile!!! Theymay break uponsudden contactwith a solidobject. Tinypipets are used topull crystals fromdrops.

Crystal mounting

Xtal is mountedin a thin-walledglass capillarytube

Xtal is mountedon a thin film ofwater in a wireloop. The loop isfixed to a metalor glass rod.

If freezing (preferred)

eucentric goniometer head(made by Nonius)

If not freezing

Must freeze immediately or filmwill dry out.!

Mounted xtal is attachedto a goniometer head forprecise adjustment.

wax

Crystal must be kept at proper humidityand temperature!! Very fragile!

Low-melting hard wax isused to ‘glue’ the rod orcapillary here.

Small wrenches fit here,here, here and here.

Why freeze?

Essentially eliminates X-ray damage to crystal.Crystals do not decay during data collection.

Why not?

Cryo equipment is expensive.

Ice crystals may form if freezing is not done properly,ruining data.

wire-loop crystal catcher

Crystals must be flash frozen

Water must be frozen to < –70°C very fast to prevent theformation of hexagonal ice. Water glass forms.

How? Crystals, mounted on loops, are flash frozen bydipping in liquid propane or freon at –70°, or by instantexposure to N2 gas at –70°C.

hexagonal ice

...to prevent glass->ice transition

Centering the crystal in thebeam

“machinecenter” is theintersection ofthe beam andthe twogoniostatrotation axes.Must be set bymanufacturer!

xrays

To place crystal at machinecenter, rotate ω and κ andwatch the crystal. If it movesfrom side to side, it is offcenter.

If it is off-center, we adjustthe screws on the goniometerhead.

whoops it’s off center. Fix it!

Aligning crystal lattice with the beam.Rotate the crystal until the zero-layer disappears and the1-layer is centered on the beam.

This

is w

here

the a

* ax

is is

poin

ted

h=1h=0h=-1 h=1

misaligned aligned

beam is here

Concentric circles around beammeans axis is aligned with beam.