Crystal Manipulation for Data Collection at Low Temperature

Crystal Manipulation for Data Collection at Low Temperature

Sean Parkin - Department of Chemistry, University of Kentucky

• Benefits of low temperature• Crystal treatment prior to cooling• Cooling methods and cryogens• Crystal mounting etc.• Potential problems• Annealing methods• When nothing seems to work …

Benefits of Low Temperature

Reduced radiation damagei) Primary – dose dependentii) Secondary – time dependentiii) Thermal damage

Decreased thermal motion (& disorder)iv) Improved resolution limit v) Possibility of disorder resolution vi) Sharper electron density

Increased crystal lifetime

Full data collection for most structurescan be done on one single crystal.

Haas & Rossmann (1970) Acta Cryst. B26, 998–1004

Intensity decay from radiation damage

Teng & Moffat (2000) J. Synch. Rad. 7, 313-317

Visible damage and reduced resolution

Lysozyme diffraction at 100K before and after ~22 minutes on beamline 14-BM-C at APS

Primary damage is dose dependent

Some molecules become chemically altered so the average electron density gets smeared.

Primary radiation damage to H2O produces •OH radicals that can initiate further damage via secondary events

Low temperature has little or no effect on primary damage.

––––––––>>x-rays––––––––>>––––––––>>

Secondary damage is time (and dose) dependent

A cascade of free-radical initiated reactions destroys long-range order even more, further smearing the electron density which increasingly destroys the high resolution data

Low temperature can inhibit secondary damage

––––––––>> x-rays time––––––––>>

Thermal damage from very intense sources*

X-ray absorption dumps energy into the crystalHeating accelerates secondary damage

Non-uniform heating causes temperature gradients and stress-induced damage

A consideration on insertion device beamlines at third generation sources.

All modes of damage will compound each other

––––––––>> intense x-rays ––––––––>>

*e.g. Teng & Moffat (2000) J. Synch. Rad. 7, 313-317

Improvement of resolution

I Ioexp{-2B(sin/)2}T1 = 300 KT2 = 100 K, Bo = 5 Å2

b = 0.05 Å2K-1

assume B(T) ≈ Bo + bT

(Bo + bT1)/r12 = (Bo + bT2)/r2

2 r2 = r1 {(Bo + bT2)/(Bo + bT1)}1/2

Roughly three times as much data at 100K vs 300KHope, H. (1988) Acta Cryst. B44, 22-26.

Reduced thermal motion

General reduction in refined B values.

Dramatic reduction may indicate resolved disorder.

BPTI, main chain BPTI, side chains

Parkin & Hope (1996) Acta Cryst. D52, 18-29.

Resolved disorder in favourable cases

BPTI C-terminus, 298K BPTI C-terminus, 125K

Parkin & Hope (1996) Acta Cryst. D52, 18-29.

Other benefits:

Full datasets are usually obtained from just one crystal.For MAD data especially, systematic errors are minimized.

Crystals can be harvested and stored.Important if crystals degrade, e.g. oxidization of Se-Met.

Crystal mounting can be much less damaging to crystals… because of reduced amount of manipulation.


Small organics can bind at active sites - not good !

Prior to cooling the crystali) Find a suitable cryoprotectant

Paratone oil, mineral oil, polyfluoroethers~60% success rate

Remove surface water

oils

antifreezes

PEG < 4K : increase PEG, add other small PEGsPEG > 4K : add small PEGsMPD : increase MPD concentrationSalt: add MPD/glycerol or even more salt ?

exchange salt e.g. to sodium formaten.b. low salt requires more help than high salt

Modify surface water

Mitchell & Garman (1996) J. Appl. Cryst. 29, 584–585

Elspeth Garman’s table of minimum amounts of glycerol needed to prevent ice formation in Hampton Screen I.

These were arrived at by dilution rather than by replacement of water, so the numbers should be used with caution.

Finding a suitable cryoprotectant

GlycerolCo-crystallizationMPDEthylene glycolPEGS (≠ PEG400)OtherPEG400CombinationOilsCryosalts


Garman & Doublié (2003) Meth. Enzymol. 368, 188-216.

Structures in Acta Cryst. D57, 2001.

0

5

10

15

20

25

30

35

5-10 10-15 15-20 20-25 25-30 30-35 35+

20002001

% of Crystals

% Glycerol (V/V)


Structures in Acta Cryst D56-57, 2000-2001.

Garman & Doublié (2003) Meth. Enzymol. 368, 188-216.

Garman (1999) Acta Cryst. D55, 1641–1653


ii) Optimize concentration of antifreeze

Mitchell & Garman (1994) J. Appl. Cryst. 27, 1070–1074

Prior to cooling the crystal

The minimum required to suppress ice is not necessarily the optimum amount

iii) Introduction of antifreezeSingle or multi-step soak / washSequential partial exchange of mother liquor Dialysis

iv) Length of soak / wash Seconds to wash, minutes (hours ?) to soak

The goal has been to minimize the shock to the crystalGenerally little optimization done unless there are real problems

Garman (1999) Acta Cryst D55, 1641-1653

Prior to cooling the crystal

a) Move crystal between solutions

0

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0 1 2 3 4 5Time (minutes)

% c

ryo-

solu

tion

b) Solution pipetted onto crystal

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% c

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Cooling methods - cryogen characteristics

Stream cool

Variable temperature is easyVery controllableSingle stepEASY

Fixed temp., 77 KVery controllable

Two stepsEASY

HarderSeveral steps Variable temp. possible but trickyNot easy to control without practice

LN2 dunk

Propane dunk

Cooling methods - cryogen cooling rates

Walker, Moreno & Hope (1998) J. Appl. Cryst. 31, 954–956Teng & Moffat (1998) J. Appl. Cryst. 31, 252–257

N2 stream liquid N2 liquid propane

N2 stream liquid N2 liquid propane

Cooling methods – points to consider

i) Cooling rate is proportional to Tii) Liquid propane can be dangerous around potential

sources of ignitioniii) Due to inherent complexity, liquid propane methods are

the hardest to make reproducibleiv) Leidenfrost gas layer insulation of large objects is

insignificant with ordinary-sized crystalsv) Liquid propane has a large liquid range - constant

stirring is required for reproducibility vi) Make the process as simple as possible


Crystal mounting – tools

Pictures: Hampton Research; Bruker-Nonius; Sean Parkin

loops - homemade

loops - bought

arcs tongs

special vialsvials and holders

Crystal mounting – aqueous film removal under oil

Aqueous films clinging to the crystal can often be teased away with a needle point.

Or they may be wicked away with a wedge of pre-moistened filter paper.

Most dry oils will accept a little water so small amounts will diffuse into the oil.This may be good or it may be bad.

Crystal mounting – crystal pick up

Crystal mounting – manipulations in the dewar

Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953.

1) Pre-cool tongs, plunge crystal.

2) Clasp the mounting pin.

3) Remove the pin holder.

4) Carry it to diffractometer.

Crystal mounting - Tongs

… transfer to diffractometer takes a couple of seconds …

… open the tongs so that the cold stream blows in the gap.

Crystal mounting - Cryovials

pictures courtesy of MSC

Note the inverted axis … … means no cryogen spillage.

Crystal mounting - robots

pictures courtesy of MSC, SSRL, Bruker-AXS

Automated

Fast

Reproducible

ExpensiveMSC - Actor BruNo

SSRL


Potential problems: Control and reproducibility

Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953

Temperature versus time for a "crystal" held in stainless steel block tongs.

Warming rate is about 0.5° per second (depends on tongs)

Crystal environment should be controlled so that it is reproducible.

Potential problems: Control and reproducibility

Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953

Temperature vs time during mount / dismount

Ensure the crystal temperature is controlled throughout mounting and that it is reproducible.

mounttemp.

dismounttemp.

Pictures: Elspeth Garman (Oxford University)

Ice caused by inadequate cryoprotectant.

Solution: optimize concentration

Ice caused by snow from slushy liquid nitrogen sticking to the drop.

Solution: carefully remount from fresh cryogen, gently tease off the snow etc.

Potential problems: Ice

Pictures: (1) Sean Parkin; (2,3) Elspeth Garman (Oxford University)

More ice problems

A poorly positioned nozzle or draughts will cause snow to grow on the pin end.

This can get serious if left too long.

1 2

3

Potential problems: Ice prevention

Even in humid environments ice can be prevented without elaborate contraptions.

The important point is a well-defined geometric relationship between cold stream, mounting pin and goniometer head and to rigorously exclude draughts

• Pin design• Cold stream geometry• Turbulence• Exclude draughts

Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953.

Potential problems: Mosaic spread

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are needed to see this picture.QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

Garman, E. (1999) Acta. Cryst. D55, 1641-1653. Dauter, Z. (1999) Acta. Cryst. D55, 1703-1717.

Minimize mosaic spread to optimise data quality.

It should prove possible to approximate the mosaicity of crystals at room temperature. Therefore it helps to know what this is !

Potential problems: maximize cooling rate

Thus a plate should cool faster than a rod or a block.

1) Keep cryogen close at hand

2) Go for a large surface area to volume ratio - so small crystals have an advantage. Generally, S/V > 12mm-1

e.g. 0.4mm x 0.4mm x 0.4mm block, S/V = 15mm-1

0.5mm x 0.5mm x 0.5mm block, S/V = 10mm-1

0.4mm x 0.5mm x 0.2mm block, S/V = 19mm-1

Things to consider

Be in control throughout the experimentThe bare minimum antifreeze concentration needed to suppress ice formation is probably not the optimum amount for minimizing mosaic spread and maximizing resolution.Minimize crystal handling.Smaller crystals are easier to cool evenly.Attempt some sort of annealing (next).

Make the mounting and retrieval process as simple as possible, but not simpler

Simplicity leads to reproducibility


Annealing of macromolecular crystals

Quick and (hopefully not so) dirty approachesQuick and (hopefully not so) dirty approaches

MacromolecularCrystal Annealing

FlashAnnealing

Systematic approachesSystematic approaches

Controlledslow annealing

Controlledflash annealing

1) "Macromolecular Crystal Annealing" - procedure

Crystal is quickly removed … … placed in cryoprotectant …

… for ~ three minutes … … and then re-flash cooled.

Harp, Timm, & Bunick, (1998) Acta Cryst. D54 622-628

Increased resolution and better mosaicity for nucleosome core particle crystal after 3 minute anneal in antifreeze or oil


Macromolecular Crystal Annealing

Hansen, Harp, & Bunick, (2003) Meth. Enzymol. 368, 217-235

A flash-cooled crystal of Patatin gave diffraction to 3.7 Å clearly showing multiple lattices. On annealing it broke into two pieces. On remounting, the larger piece diffracted to ~2.3 Å.


An otherwise trashed nucleosome core particle crystal resurrected after a 3 minute anneal



2) "Flash Annealing" - procedure

Yeh & Hol (1998) Acta Cryst. D54, 479-480

The cold stream blowing over the flash cooled crystal is blocked for a short period of time (seconds) until it has thawed.

Then the obstruction is released to re-cool the crystal.

Flash cooled … … stream diverted … … re-flash cooled.

Flash annealing - results

Glycerol kinase, resolution limit ~4 Å, poor mosaic spread. Flash annealing by blocking the cold stream for 1.5 - 2 seconds three times gives ~2.8 Å resolution and better mosaic spread.

Yeh & Hol (1998) Acta Cryst. D54, 479-480

Accomplished in two ways:

Slow warming using a controllable stream heater.

Rapid warming to some pre-determined temperatureby either dynamic mixing of cold and warm streamsor by rapid switching of two cold gas streams.

Questions to be answered:

Is it a protein or a bulk water phenomenon ?

How does annealing work ?

Why does annealing work ?

What are the mechanisms of protein crystal annealing ?

Controlled annealing without thawing

3) Slow annealing: Cell volume vs temperature

V > 1200 Å3

Concanavalin A1

2) Weik et al. (2001) Acta Cryst. D57, 566-573

TcAChE (trigonal)2

TcAChE (orthorhombic)2

Crystals with channels show an abrupt volume jump at some well defined temperature.Implies that effect is in the bulk water and that there is a surface effect via the connection to the crystal surface.

1) Parkin (1993) Ph.D. Thesis, UC Davis

475000

476000

477000

478000

479000

480000

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482000

80 100 120 140 160 180 200

temperature (K)

Cell dimension changes on annealing

0.99950

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temperature (K)

chan

ge r

elat

ive

to 9

5 K

a axis

b axis

c axis

There is an abrupt jump in both the b and c axes in concanavalin A on warming from 160 to 165 K. There is no corresponding jump in a.

Parkin & Hope (2003) Acta Cryst. D59, 2228-2236

(view down c )

c axis expands protein moves waters move

(view down b )

b axis expands protein moves waters move

(view down a )

a axis constant -

waters move

Origin of annealing effects

c a

b c

a b


10 15 20 25 30 35 40 45 50 55 60

diffraction angle (degrees)

set 1

set 3

set 2

Annealing affects diffuse solvent diffraction

Overall background for concanavalin A diffraction is reduced after annealing. It is also a bit smoother.

Still don’t know what the likely mechanism of annealing is.


4) Flash annealing without thawing

Rapid adjustment of warm and cold gas flows onto the crystal.Can be tricky

With two low-temperature machines we can rapidly switch

cold streams.

Much easierKriminski, Caylor, Nonato, Finkelstein, & Thorne Acta Cryst. (2002), D58, 459-471 Parkin (2002) unpublished

Flash annealing seen by in-situ X-ray imaging

At room temp. the whole crystal is in the diffracting position over a very small angular range.

After flash cooling, the mosaic spread is much worse and the resolution limit was severely degraded to 4.3 Å.

After 25 s controlled anneal at 250 K, resolution limit is 2.4 Å, mosaic spread generally not as good as pre-cool value.

Kriminski, Caylor, Nonato, Finkelstein, & Thorne Acta Cryst. (2002), D58, 459-471

What happens to the mosaic structure ?

On flash cooling, individual domains have a mosaic spread similar to that of the whole crystal. After annealing, small domains have much narrower mosaic spread, but are themselves somewhat mis-aligned.


What happens to the water ?

a) The distribution of water is fairly uniform in solvent regions of fresh crystals at room temperature.

b) During flash cooling, water is squeezed out of small domains and collects in the regions surrounding the domains.

c) Which leaves the domains somewhat more mis-aligned.

Annealing likely gives a partial fix by increasing order within domains and by reducing the spread of lattice spacings within a crystal.


i) Consider annealing if diffraction is uncharacteristically poor after flash cooling.

ii) Try the quick methods first. MCA appears to be more general.

iii) For MCA, the crystal must be stable in its cryoprotectant.

iv) Size may be a less important factor than shape. Thin crystals may be better suited to flash annealing.

v) In MCA, a full three minutes may not be needed. Longer times appear less likely to yield optimum results.

vi) Multiple cycles of MCA are not necessary and may be undesirable. For flash annealing, multiple cycles may be required.

Annealing - General comments

Hansen, Harp, & Bunick, (2003) Meth. Enzymol. 368, 217-235


When nothing seems to work (in no particular order):

Does it diffract at room temperature ?

Try other cryoprotectants

Try more than once.

Vary time and temperature of crystal handling steps

Match antifreeze to the system

Exchange buffers etc.

Attempt annealing.

Does the crystal diffract at room temperature ?

Photograph: Elspeth Garman (Oxford University)Photograph: Elspeth Garman (Oxford University)

Capillary scheme lifted from “Practical Protein Crystallography” by Duncan McRee

Try more than once.

Picture courtesy Elspeth Garman, after Schneider, Bravo & Hansen

Match antifreeze to the system - Osmotic Pressures:

1) Find osmotic pressure of mother liquor in the CRC Handbook of Chemistry and Physics (section D232) 11th column, O (Os/Kg)

2) Find osmotic pressure of your antifreeze.

3) Modify the concentration in mother liquor to minimize the change in osmotic pressure.

Osmotic shock: Water will either be pumped into the crystal or sucked out of the crystal. Either one may cause cracks, increase mosaicity, lower resolution etc. All bad.

Rapid transfers between solutions will give greatest shock but will minimize the time over which damage could occur.

Experiment !

Osmotic pressure matching - Elspeth’s worked example.

Mother Liquor: Osmolarity (Os/Kg)2.0 M NaCl50mM pH 7.8 Tris HCl 3.95

Cryoprotectant:20% glycerol 2.90

Difference here is 1.05, so the plan is to alter the cryoprotectant sothat it matches. From the CRC Handbook, 0.55M NaCl exerts an osmotic pressure of 1.05 Os/Kg.

Hence try 0.55M NaCl, 20% glycerol in 50mM pH 7.8 Tris HCl

Advantages and disadvantages of low temperature work.

FOR:

Reduced radiation damage

Gentler mounting

Lower backgrounds

Higher resolution

Fewer crystals

Transportation is easy

Harvest crystals at their peak.

AGAINST:

Expense (money)

Expense (time)

Mosaic spread increase

Documents

Crystal Manipulation for Data Collection at Low Temperature