A4

1
Should we be doing more crystallization by the
microbatch method? Patrick Shaw
Stewart Imperial College, London Professor
David M. Blow, Patrick Shaw Stewart, Dennis
Maeder, Naomi Chayen Douglas Instruments Limited
(near Oxford, UK) Peter Baldock, Patrick Shaw
Stewart, Vaughan Mills, James Smith
2

1. What is the microbatch method?
2. Hardware and dispensing routines
3. Phase diagrams
4. Comparisons of microbatch and vapor diffusion
5. Case studies
6. Harvesting crystals
7. Experimental design

3

1. What is the microbatch method?
2. Hardware and dispensing routines
3. Phase diagrams
4. Comparisons of microbatch and vapor diffusion
5. Case studies
6. Harvesting crystals
7. Experimental design

4
What is the microbatch method?

• Crystallization in small drops under oil

5
What is the microbatch method?

• Crystallization in small drops under oil
• 100 100 nl to 11 µl

6
What is the microbatch method?

• Crystallization in small drops under oil
• 100 100 nl to 11 µl
• The oil prevents evaporation

7
Why is microbatch a good idea?
8
Why is microbatch a good idea?

1. Easy

9
Why is microbatch a good idea?

1. Easy
2. Gives better crystals in many cases especially
   in screening

10
Why is microbatch a good idea?

1. Easy
2. Gives better crystals in many cases especially
   in screening
3. It doesnt matter if the security guard at the
   airport puts it through the x-ray machine upside
   down

11
Why is microbatch a good idea?

• Easy
• Gives better crystals in many cases especially
  in screening
• It doesnt matter if the security guard at the
  airport puts it through the x-ray machine upside
  down
• Cheap!

12
Microbatch crystallization

Volume of well - 12 microlitres
13
Microbatch crystallization

Volume of drop - 0.2 to 2 microlitres
14
Microbatch crystallization

(2-bore) microtip
Oil
Sample
15
Microbatch crystallization

16
Microbatch crystallization

17
Microbatch optimization print out
Row 1
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
Row 2
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
Row 3
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
Row 4
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
18
Microbatch optimization print out
Row 1
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
Row 2
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
Row 3
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
Row 4
50 mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3 M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100 Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
95 PEG 600 dyed red 0.12 0.11 0.1 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
19
ORYX 6 crystallization system
20
Liquid-handling channel
Motorized Hamilton gas-tight syringe (water)
X 5
to microtip
21
ORYX 6 crystallization system
22
Large-volume tip for oil

23
New Chassis design

24
New Chassis design

25
Left-hand tip
2-bore Microtip screening
5-bore Microtip optimization
26
End of a 5-bore microtip

0.15 mm
0.9 mm
27
Sitting Drop
28
Sitting Drop - preparation

• Use a 2-bore microtip

29
Sitting Drop - preparation

• Use a 2-bore microtip
• Start with both bores full of water

30
Sitting Drop - preparation

• Use a 2-bore microtip
• Start with both bores full of water
• Suck up 1µl of air into both channels

Air bubbles
31
Sitting Drop - preparation
Air bubble

• Use a 2-bore microtip
• Start with both bores full of water
• Suck up 1µl of air into both channels
• Suck up protein required for experiment 0.25 µl

Protein slug
32
Sitting Drop dispensing cycle

• Rinse in reservoir

(1)
33
Sitting Drop dispensing cycle

• Rinse in reservoir
• Move sideways and pick up clean solution

(1)
(2)
34
Sitting Drop dispensing cycle

• Rinse in reservoir
• Move sideways and pick up clean solution
• Dispense solution and protein

(3)
(1)
(2)
35
Microbatch screening
36
Microbatch screening dispensing cycle
Target plate
Screening solutions
37
Microbatch screening dispensing cycle

• Pick up screening solution

(1)
38
Microbatch screening dispensing cycle

• Pick up screening solution
• Transfer to microbatch drop

(1)
(2)
39
Microbatch screening dispensing cycle

• Pick up screening solution
• Transfer to microbatch drop oil

(1)
(2)
oil
40
Microbatch screening dispensing cycle

• Pick up screening solution
• Transfer to microbatch drop oil
• Rinse

(1)
(2)
(3)
oil
41
Microbatch optimization
42
Microbatch optimization dispensing cycle

• Dispense five solutions together

(1)
43
Microbatch optimization dispensing cycle

• Dispense five solutions together
• Oil

(1)
oil
44
Microbatch optimization dispensing cycle

• Dispense five solutions together
• Oil

(1)
oil
45
Microbatch optimization dispensing cycle

• Dispense five solutions together
• Oil

(1)
oil
46
Central Composite design

47
Phase diagram of a protein

precipitate
Protein
clear
Precipitant
48
Phase diagram of a protein

precipitate
nucleation
Protein
clear
Precipitant
49
Phase diagram of a protein

50
Phase diagram of a protein

p
n
Protein
m.z.
Vapor diffusion
c
Precipitant
51
Phase diagram of a protein

p
n
Microbatch
Protein
m.z.
v.d.
c
Precipitant
52
Phase diagram of a protein

p
n
M.B.(paraffin)
Protein
m.z.
v.d..
M.B.(par./si.)
c
Precipitant
53
Phase diagram of a protein

p
n
M.B.(paraffin) OPTIMIZATION
Protein
m.z.
v.d.
M.B.(par./si.) SCREENING
Precipitant
54
What of protein should you use?
Microbatch with Si. / Par.
n
Protein
m.z.
Precipitant saturated
Precipitant
55
What of protein should you use?
Microbatch with Si. / Par.
n
Protein
Protein stock
m.z.
50
Precipitant saturated
Precipitant stock
Precipitant
56
What of protein should you use?
Microbatch with Si. / Par.
n
Protein
Protein stock
m.z.
66
50
Precipitant saturated
Precipitant stock
Precipitant
57
Screening studies comparing microbatch with
vapor diffusion
    Proteins Conditions MB VD Extra hits for MB   Extra hits for MB Unique to MB Unique to VD
1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5 17 15

P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A
comparison of microbatch and vapour diffusion for
initial screening of crystallization conditions.
Journal of Crystal Growth. 168 (1996), pp 170-174
or http//www.douglas.co.uk/rep2.htm
58
Screening studies comparing microbatch with
vapor diffusion
    Proteins Conditions MB VD Extra hits for MB   Extra hits for MB Unique to MB Unique to VD
1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5 17 15
2000 D'Arcy et al. Hoffman-La Roche 10 48 104 62 42 68    

P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A
comparison of microbatch and vapour diffusion for
initial screening of crystallization conditions.
Journal of Crystal Growth. 168 (1996), pp 170-174
or http//www.douglas.co.uk/rep2.htm A. DArcy,
G.E. Dale, M. Stihle, B. DArcy. Results
reported at the 8th International Conference on
the Crystallization of Biological Macromolecules,
May 18, 2000.
59
Screening studies comparing microbatch with
vapor diffusion
    Proteins Conditions MB VD Extra hits for MB   Extra hits for MB Unique to MB Unique to VD
1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5 17 15
2000 D'Arcy et al. Hoffman-La Roche 10 48 104 62 42 68    
2001 Noordeen et al. Novartis Pharma 8 48 - 576 145 153 -8 -5 95 103

P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A
comparison of microbatch and vapour diffusion for
initial screening of crystallization conditions.
Journal of Crystal Growth. 168 (1996), pp 170-174
or http//www.douglas.co.uk/rep2.htm A. DArcy,
G.E. Dale, M. Stihle, B. DArcy. Results
reported at the 8th International Conference on
the Crystallization of Biological Macromolecules,
May 18, 2000. N. Noordeen and S. Cowan-Jacob.
Novartis Pharma AG. http//www.hamptonresearch.com
/stuff/ppt_files/P6.ppt
60
Screening studies comparing microbatch with
vapor diffusion
    Proteins Conditions MB VD Extra hits for MB   Extra hits for MB Unique to MB Unique to VD
1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5 17 15
2000 D'Arcy et al. Hoffman-La Roche 10 48 104 62 42 68    
2001 Noordeen et al. Novartis Pharma 8 48 - 576 145 153 -8 -5 95 103
Sugahara SPring8 6 288 100 84 16 19    
 

P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A
comparison of microbatch and vapour diffusion for
initial screening of crystallization conditions.
Journal of Crystal Growth. 168 (1996), pp 170-174
or http//www.douglas.co.uk/rep2.htm A. DArcy,
G.E. Dale, M. Stihle, B. DArcy. Results
reported at the 8th International Conference on
the Crystallization of Biological Macromolecules,
May 18, 2000. N. Noordeen and S. Cowan-Jacob.
Novartis Pharma AG. http//www.hamptonresearch.com
/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken
Harima Institute, SPring8. Personal
communication.
61
Screening studies comparing microbatch with
vapor diffusion
    Proteins Conditions MB VD Extra hits for MB   Extra hits for MB Unique to MB Unique to VD
1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5 17 15
2000 D'Arcy et al. Hoffman-La Roche 10 48 104 62 42 68    
2001 Noordeen et al. Novartis Pharma 8 48 - 576 145 153 -8 -5 95 103
Sugahara SPring8 6 288 100 84 16 19    
TOTAL    30   392 340 52  15    

P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A
comparison of microbatch and vapour diffusion for
initial screening of crystallization conditions.
Journal of Crystal Growth. 168 (1996), pp 170-174
or http//www.douglas.co.uk/rep2.htm A. DArcy,
G.E. Dale, M. Stihle, B. DArcy. Results
reported at the 8th International Conference on
the Crystallization of Biological Macromolecules,
May 18, 2000. N. Noordeen and S. Cowan-Jacob.
Novartis Pharma AG. http//www.hamptonresearch.com
/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken
Harima Institute, SPring8. Personal
communication.
62
OPTIMIZATION about 5050

• In microbatch, there tends to be more
  precipitation initially this may result in more
  nucleation

63
OPTIMIZATION about 5050

• In microbatch, there tends to be more
  precipitation initially this may result in more
  nucleation
• In a survey of about 30 protein samples at
  Imperial College, London, the best data was
  collected from MB in 50 of cases

64
OPTIMIZATION about 5050

• In microbatch, there tends to be more
  precipitation initially this may result in more
  nucleation
• In a survey of about 30 protein samples at
  Imperial College, London, the best data was
  collected from MB in 50 of cases
• Lesley Haire (NIMR, London) told me that out of
  12 structures solved in the last few years, 5
  relied on microbatch

65
OPTIMIZATION about 5050
Vapor diffusion
Microbatch

From DArcy et al. A novel approach to
crystallising proteins under oil. Journal of
Crystal Growth 168 (1996) 175-180.
66
Crystals obtained at 4ºC(Lesley Haire, Imperial
College)

67
Crystals nucleated for 1 hr 4ºC, then grown at
18ºC

68
Case Study 2Use of microseeding
Yaakov Korkhin and Artem Evdokimov, Weizmann
Institute of Science, Israel A newly isolated
alcohol dehydrogenase from a thermophile was
crystallized with PEG 4000, pH 5.5 - 8.6

• VD crystals grew very rapidly and were poorly
  formed

• MB crystals were initially similar

69
1. Determination of phase diagram

p
Protein
m.z.
Precipitant
70
A few good quality crystals were obtained

71
Edge of nucleation 16 PEG
Protein
PEG 4K
72
2. Microseeding was used

1. A well-formed crystal was broken up in 15.5 PEG
2. The mixture was spun
3. A series of dilutions was set up using the
   supernatant (11000 worked best)

73
Reservoir 16.5
Droplet 15.5
Protein
PEG 4K
74
Reproducible good quality crystals wereobtained
with microseeding. Crystals diffracted to 2Å

75
Exactly the same conditions but with no
seeding solution - gave poor crystals

76
Vapor batch crystallization using volatile
organic solvents

• Lesley Haire
• Division of Protein Structure,
• National Institute for Medical Research,
• The Ridgeway, Mill Hill, London NW7 1AA, UK
• Winner of the competition for the
• Best Use of the Douglas Vapor Batch Plate
• First round - January 2005

77
Crystallisation of NTD ofN-MLV capsid

• Crystals were grown from hanging drops - Hampton
  Crystal Screen no.40, 20 PEG 4000, 20 v/v
  isopropanol, 0.1M Nacitrate pH 5.6 20mg/ml
  protein in the drops
• Major problem - harvesting the crystals in the
  presence of isopropanol.
• The crystals disintegrated as soon as the
  coverslip was opened.

78
Attempts to overcome the problem

• Using sitting drops,
• oil over the drops,
• and handling crystals using constant humidity
  were only partially successful.
• In microbatch experiments under oil, crystals
  were not stable and dissolved after a couple of
  days.
• Crystals that were X-rayed had high mosaicity and
  could not be used for structure solution.

79
Vapor Batch trays (Douglas Instruments)
80

• Procedure
• Droplets (2?l) dispensed under a mixture of
  silicone/ paraffin oil using IMPAX 1-5
  crystallisation robot.
• A 6x4 spreadsheet was set up with XSTEP software
  varying protein, 16-22 mg/ml PEG 3350, 13-16
  all wells had 0.1M sodium citrate pH 5.6.
• 10 isopropanol was pipetted into the trays
  moat and the drops equilibrated overnight at
  18?C.
• Next day, the 10 isopropanol solution was
  replaced by 20 isopropanol

81

• This method was used to grow crystals of NTD
  N-MLV capsid protein
• Crystals appeared after a couple of days.
• Typically they were harvested and frozen after 10
  days.
• Crystals were very stable in drops for at least 6
  months.
• Diffraction to 1.9Å with low mosaicity.
• Crystals did not grow in the controls without
  isopropanol in the moat.
• Capsid protein was provided by Nehar Mortuza

82
NTD N-tropic MLV- capsid protein
G. B. Mortuza, L. F. Haire, A. Stevens, S. J.
Smerdon, J. P. Stoye I. A. Taylor. Nature
(2004) 431 481-485.
83
Using vapor batch in screening Low ionic
strength PEG screens Vary pH and PEG
concentration /- isopropanol or other volatile
organic in the moat. High salt screens Use
AmS04 or P04, set up duplicate trays, /-10
isopropanol in the moat. The same principle
could be used to test isopropanol or any other
volatile additive with a selected screen
dispensed in VB trays.
84
Crystals grown by VB with isopropanol
catalase
1918 H1

Low ionic strength PEG screen, Sigma
85
Advantages of vapor batch cf. vapor diffusion

• Improved crystal stability
• Easier crystal handling
• Better diffraction from crystals grown under
  paraffin/silicone oil mixture.

86
Harvesting Crystals from Microbatch
James Liu - University of Georgia Jeroen Mesters
- University of Luebeck
87
Harvesting Crystals from Microbatch

• James Liu B.C. Wangs group, University of
  Georgia
• High-throughput crystallization for structural
  genomics

88
Harvesting Crystals from Microbatch

• James Liu
• Microbatch is easier because the oil prevents
  evaporation - you can work slowly!
• You can loop straight out of the droplet through
  the oil.
• James record he mounted 98 crystals in one
  day!

89
Harvesting Crystals from Microbatch

• Digression
• University of Georgia is unusual it uses sitting
  drop for screening and microbatch for
  optimization.
• This reduces the solution volumes needed and
  solutions can be reused many times.

90
Harvesting Crystals from Microbatch
Jeroen Mesters

1. Use a loop with a bent handle.
2. Make sure the crystal fits the loop well or the
   oil will drag it off.

91
Multivariate experimental design

• Almost all protein crystallization experiments
  have at least 4 parameters
• Protein concentration
• Precipitant concentration
• pH
• Temperature
• Additive ? .

92
Central Composite design

93
Box-Behnken design

94
The autodesign function of XSTEP .

95
. automatically fills a spreadsheet

96
. and XSTEP executes it.
97

• The Biblical Zoo in Jerusalem

98
ORYX (arabian)
99
Imperial College, London Professor David
M. Blow, Patrick Shaw Stewart, Dennis Maeder,
Naomi Chayen Douglas Instruments Limited (near
Oxford, UK) Peter Baldock, Patrick Shaw Stewart,
James Smith, Vaughan Mills
100
And finally - Yaakov showed me
101
What Yaakov saw
102
How can we do vapor diffusion as easily as
microbatch?

103
Vapor Diffusion effect demonstrated by increasing
reservoir concentration

Number of crystals
? 0.5 M AS
? 1.0 M AS
Days
104
Experimental Design Steps
Step 1. Primary Screen. Approx.
30-dimensional search. E.g. Sparse Matrix or
Incomplete Factorial Step 2. Targeted
Screen Approx. 10-dimensional search. E.g.
Incomplete factorial or Crystool
optimization Step 3. Multidimensional
Grid Approx. 4-dimensional search. E.g.
Central Composite, Box Behnken - XSTEP
Autodesign Step 4. 2-D Grid Approx.
2-dimensional search. E.g. XSTEP grids.