Douglas Instruments 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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Experimental design for high throughput protein crystallization Patrick Shaw Stewart
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 Presentation
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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 )
• 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
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:
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:
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. 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. 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:
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. 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:
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 ? …………….