experimental design for high throughput protein crystallization patrick shaw stewart
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Experimental design for high throughput protein crystallization Patrick Shaw Stewart Douglas Instruments Limited (near Oxford, UK): ( A copy of this file can be found at http://www.douglas.co.uk/resrep.htm ). Experimental design for high throughput protein crystallization. - PowerPoint PPT PresentationTRANSCRIPT
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Microbatch seminar- slide 1
Experimental design for high throughput protein
crystallization
Patrick Shaw Stewart
Douglas Instruments Limited (near Oxford, UK):( A copy of this file can be found at http://www.douglas.co.uk/resrep.htm )
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Microbatch seminar- slide 2
Experimental design for high throughput protein crystallization
• Largely the same as for low throughput experimental design, but:
• Good design is more important.
• Don’t waste time thinking – do the thinking first
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Microbatch seminar- slide 3
1. Degree of automation
2. Crystallization methods (with phase diagrams)
3. Experimental design – steps of protein crystallization projects
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Microbatch seminar- slide 4
1. Degree of automation
2. Crystallization methods (with phase diagrams)
3. Experimental design – steps of protein crystallization projects
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Microbatch seminar- slide 5
Automation: don’t over-automate!Per year
Per week
Per day
WorthAutomating? Assumptions
Structures 70 1.4 Target: structures per year 70
Purified proteins 9.8 2Purified proteins/structure
(SPINE) 7Screening:
Plates 38 8 Screens (96 wells) per protein 4(Reservoirs to be dispensed) /
12 64 NO(Drops to be dispensed) * 2 1536 YES
Optimization: Optimizations per structure 1Plates 4.2 0.8 Plates per optimization 3
Reservoirs to be dispensed 81
YES, but it’s not easy (use
microbatch?)(Drops to be dispensed) * 2 161 Yes
Crystal observation:Plates to move to imager 44 9 NO
Images to collect and view 6789 YES Images collected per drop 4Crystals to mount 17 3 NO Crystals per structure (UGA) 12
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Microbatch seminar- slide 6
Automation: don’t over-automate!
• Recovery from errors can be very time-consuming
• Avoid long chains of automatic systems
• Use human buffer zones e.g. move plates by hand
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Microbatch seminar- slide 7
1. Degree of automation
2. Crystallization methods (with phase diagrams)
3. Experimental design – steps of protein crystallization projects
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Microbatch seminar- slide 8
Vapor diffusion
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Microbatch seminar- slide 9
Phase diagram of a protein
[Protein]
[Precipitant]
precipitation
nucleation
metastable zone
clear
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Microbatch seminar- slide 10
Thermodynamic processes which develop so slowly as to allow each intermediate step to be an equilibrium state are said to be reversible processes.
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Microbatch seminar- slide 11
Phase diagram of a protein
[Protein]
[Precipitant]
pn
m.z. Vapor diffusion
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Microbatch seminar- slide 12
Vapor diffusion
• Works well
• Gentle – drop is concentrated AFTER mixing
• Doesn’t suit all proteins
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Microbatch seminar- slide 13
Dialysis
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Microbatch seminar- slide 14
Phase diagram of a protein
[Protein]
[Precipitant]
pn
m.z.
Dialysis
V.D.
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Microbatch seminar- slide 15
Dialysis
• Gives a lot of control
• You have to be patient
• Not easy to automate
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Microbatch seminar- slide 16
Microbatch
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Microbatch seminar- slide 17
Phase diagram of a protein
[Protein]
[Precipitant]
pn
m.z.
M.B (paraffin)
Dialysis
V.D.
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Microbatch seminar- slide 18
Phase diagram of a protein
[Protein]
[Precipitant]
pn
m.z.
M.B (paraffin)
M.B. (Si / paraffin)
Dialysis
V.D.
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Microbatch seminar- slide 19
Phase diagram of a protein
[Protein]
[Precipitant]
pn
m.z.
M.B (p) OPTIMIZATION
M.B. (Si/p) SCREENING
Dialysis
V.D.
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Microbatch seminar- slide 20
Microbatch
• Simple and cheap
• Versatile – screening / optimization, different oils, additives, volatile reagents (ethanol, iso-propanol etc.)
• Suits some proteins very well
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Microbatch seminar- slide 21
Counter-diffusion
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Microbatch seminar- slide 22
Phase diagram of a protein
[Protein]
[Precipitant]
pn
m.z.
M.B (paraffin)
M.B. (Si/paraffin)
Dialysis
Counter-diffusion
V.D.
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Microbatch seminar- slide 23
• Arguably the BEST physical method of crystallization
• Gives “self-selection” of crystallization conditions
• Not easy to automate, but quite easy to set up by hand
• 18 examples in the PDB
Counter-diffusion
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Microbatch seminar- slide 24
• Arguably the BEST physical method of crystallization
• Gives “self-selection” of crystallization conditions
• Not easy to automate, but quite easy to set up by hand
• 18 8 examples in the PDB
Counter-diffusion
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Microbatch seminar- slide 25
What % volume of protein should you use?
100 nl + 100 nl ?
200 nl + 100 nl ?
1 µl + 1 µl ?
2 µl + 1 µl ?
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Microbatch seminar- slide 26
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Precipitant saturated
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Microbatch seminar- slide 27
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Protein stock
Precipitant stock
Precipitant saturated
50%
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Microbatch seminar- slide 28
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Protein stock
Precipitant stock
Precipitant saturated
50%66%
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Microbatch seminar- slide 29
What % volume of protein should you use?
Increasing the proportion of protein in the drop:
1. Reduces the chance of salt crystals
2. Facilitates scaling up from nanodrops (personal communication, Heather Ringrose, Pfizer)• Use e.g. 0.2 µl (protein) + 0.1 µl (reservoir soln.)• This scales up to 1 + 1 µl (protein may be lost by
denaturation in small samples, and small samples equilibrate faster)
• Generally, data mining suggests that you should increase the salt in larger drops
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Microbatch seminar- slide 30
1. Degree of automation
2. Crystallization methods (with phase diagrams)
3. Experimental design – steps of protein crystallization projects
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Microbatch seminar- slide 31
Conventional Approach
1. Screening – get first crystals
2. Optimization – improve crystals
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Microbatch seminar- slide 32
Experimental Design Steps
Step 1. “Primary Screen.” Approx. 60-dimensional search.
Step 2. “Targeted Screen” Approx. 12-dimensional search.
Step 3. “Multidimensional Grid” Approx. 5-dimensional search.
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Microbatch seminar- slide 33
Experimental Design StepsStep 0. “Prescreen” to find precipitation points1-dimensional search.
Step 1. “Primary Screen.” Approx. 60-dimensional search.
Step 2. “Targeted Screen” Approx. 12-dimensional search.
Step 3. “Multidimensional Grid” Approx. 5-dimensional search.
Step 4. “2-D Grid” 2-dimensional search.
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Microbatch seminar- slide 34
Experimental Design StepsStep 0. “Prescreen” to find precipitation points1-dimensional search. E.g. Pre-crystallization assay,
Pre Screening Assay, Footprint Screen
• Use to adjust protein concentration• Automation is available
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Microbatch seminar- slide 35
Experimental Design Steps
Step 1. “Primary Screen.” Approx. 60-dimensional search. E.g. Sparse Matrix
• Many robotic systems are available• Use pre-mixed solutions
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Microbatch seminar- slide 36
Experimental Design Steps
Step 2. “Targeted Screen” Approx. 12-dimensional search. 1. Additive approach2. De novo approach
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Microbatch seminar- slide 37
Step 3: “Targeted Screen”1. Additive approache.g. You get a hit in Jancarik and Kim screen = 0.2M Mg formate You make a targeted screen by adding 10 % of a second screen to the successful condition:
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Microbatch seminar- slide 38
Step 3: “Targeted Screen”1. Additive approache.g. You get a hit in Jancarik and Kim screen = 0.2M Mg formate You make a targeted screen by adding a second screen to the successful condition:
2.1 0.18M Mg formate + 0.1M Na acetate pH 4.62.2 0.18M Mg formate + 0.1M Na citrate pH 6.52.3 0.18M Mg formate + 4% w/v PEG 80002.4 0.18M Mg formate + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
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Microbatch seminar- slide 39
Step 3: “Targeted Screen”1. Additive approache.g. You get a hit in Jancarik and Kim screen = 0.2M Mg formate You make a targeted screen by adding a second screen to the successful condition:
2.1 0.18M Mg formate + 0.1M Na acetate pH 4.62.2 0.18M Mg formate + 0.1M Na citrate pH 6.52.3 0.18M Mg formate + 4% w/v PEG 80002.4 0.18M Mg formate + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
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Microbatch seminar- slide 40
Step 3: “Targeted Screen”1. Additive approache.g. You get a hit in Jancarik and Kim screen = 0.2M Mg formate You make a targeted screen by adding a second screen to the successful condition:
2.1 0.18M Mg formate + 0.1M Na acetate pH 4.62.2 0.18M Mg formate + 0.1M Na citrate pH 6.52.3 0.18M Mg formate + 4% w/v PEG 80002.4 0.18M Mg formate + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
2. De novo approache.g. You get a hit in Jancarik and Kim screen = 30% w/v PEG 1500 You mix up a targeted screen by adding a second screen to the successful condition:
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Microbatch seminar- slide 41
Step 3: “Targeted Screen”1. Additive approache.g. You get a hit in Jancarik and Kim screen = 0.2M Mg formate You make a targeted screen by adding a second screen to the successful condition:
2.1 0.18M Mg formate + 0.1M Na acetate pH 4.62.2 0.18M Mg formate + 0.1M Na citrate pH 6.52.3 0.18M Mg formate + 4% w/v PEG 80002.4 0.18M Mg formate + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
2. De novo approache.g. You get a hit in Jancarik and Kim screen = 30% w/v PEG 1500 You mix up a targeted screen by adding a second screen to the successful condition:
3.1 30% v/v PEG 600 3.2 20% w/v PEG 40003.3 25% w/v PEG 1500 + 0.1M Na acetate pH 4.63.4 20% w/v PEG 4000 + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
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Microbatch seminar- slide 42
Step 3: “Targeted Screen”1. Additive approache.g. You get a hit in Jancarik and Kim screen = 0.2M Mg formate You make a targeted screen by adding a second screen to the successful condition:
2.1 0.18M Mg formate + 0.1M Na acetate pH 4.62.2 0.18M Mg formate + 0.1M Na citrate pH 6.52.3 0.18M Mg formate + 4% w/v PEG 80002.4 0.18M Mg formate + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
2. De novo approache.g. You get a hit in Jancarik and Kim screen = 30% w/v PEG 1500 You mix up a targeted screen by adding a second screen to the successful condition:
3.1 30% v/v PEG 600 3.2 20% w/v PEG 40003.3 25% w/v PEG 1500 + 0.1M Na acetate pH 4.63.4 20% w/v PEG 4000 + 4% w/v 2-methyl-2,4-
pentanediol ……………. etc.
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Microbatch seminar- slide 43
Step 3: “Targeted Screen”1. Additive approach• Easy to set up / automate• Some limits on where you can go• Doesn’t greatly reduce the number of variables that
you have to deal with• E.g. Nextal’s Optimizer
2.De novo approach• Difficult and slow to automate• All areas of crystallization space are accessible• Contributes to the reduction of the number of
variables• E.g. Matrix Maker, Pick & Mix software • Allows “reshuffling” of ingredients in separate hits
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Microbatch seminar- slide 44
Experimental Design Steps
Step 3. “Multidimensional Grid” Approx. 5-dimensional search. E.g. Central Composite, Box Behnken, XSTEP Autodesign
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Microbatch seminar- slide 45
Multivariate experimental design
Almost all protein crystallization experiments have at least 4 parameters:
1. Protein concentration2. Precipitant concentration3. pH4. Temperature5. Additive ? …………….
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Microbatch seminar- slide 46
Central Composite design
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Microbatch seminar- slide 47
Box-Behnken design
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Microbatch seminar- slide 48
The Autodesign function of XSTEP ….
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Microbatch seminar- slide 49
…. automatically fills a “spreadsheet” …
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Microbatch seminar- slide 50
…. and XSTEP executes it.
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Microbatch seminar- slide 51
Experimental Design Steps
Step 4. “2-D Grid” Approx. 2-dimensional search. E.g. XSTEP grids, manual experiments
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Microbatch seminar- slide 52
Xstep Optimization 2-d grid
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Microbatch seminar- slide 53
2-Dimensional grids
• Probably not really needed• Arguably it is good to make very small
changes for production plates (to make crystals for data collection)
• [Precipitant] vs [protein] or [precipitant] vs pH
• Easy to set up and interpret
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Microbatch seminar- slide 54
Experimental Design StepsStep 0. “Prescreen” to find precipitation points1-dimensional search.
Step 1. “Primary Screen.” Approx. 60-dimensional search.
Step 2. “Targeted Screen” Approx. 12-dimensional search.
Step 3. “Multidimensional Grid” Approx. 5-dimensional search.
Step 4. “2-D Grid” 2-dimensional search.
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Microbatch seminar- slide 55
Reducing the dimensions that must be considered60
24
12
5
2
TIME
Number of dim
ensions that you are thinking
about
60 dimensions (ingredients)
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Microbatch seminar- slide 56
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
?
TIME
Number of dim
ensions that you are thinking
about
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Microbatch seminar- slide 57
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
?
TIME
Number of dim
ensions that you are thinking
about
Thinking ……
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Microbatch seminar- slide 58
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
?
TIME
Number of dim
ensions that you are thinking
about
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Microbatch seminar- slide 59
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
? ?
Additve-type targeted screen
TIME
Number of dim
ensions that you are thinking
about
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Microbatch seminar- slide 60
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
? ?
Additve-type targeted screen
De novo targeted
screen
TIME
Number of dim
ensions that you are thinking
about
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Microbatch seminar- slide 61
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
? ?
?
Additve-type targeted screen
De novo targeted
screen Multi-d screen
TIME
Number of dim
ensions that you are thinking
about
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Microbatch seminar- slide 62
Reducing the dimensions that must be considered60
24
12
5
2
Primary screen
? ?
? ?
Additve-type targeted screen
De novo targeted
screen Multi-d screen
2-d grid
TIME
Number of dim
ensions that you are thinking
about
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Microbatch seminar- slide 63
Finally ….
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Microbatch seminar- slide 64
Oryx (arabian)
The Biblical Zoo in Jerusalem