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									                                                                                                             Douglas Instruments
                                           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 )




Microbatch seminar- slide 1
           Experimental design for high throughput




                                                                                                 Douglas Instruments
                   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




Microbatch seminar- slide 2
                                                                               Douglas Instruments
          1. Degree of automation

          2. Crystallization methods (with phase diagrams)

          3. Experimental design ‟ steps of protein crystallization projects




Microbatch seminar- slide 3
                                                                               Douglas Instruments
          1. Degree of automation

          2. Crystallization methods (with phase diagrams)

          3. Experimental design ‟ steps of protein crystallization projects




Microbatch seminar- slide 4
                          Automation: don’t over-automate!




                                                                                                                                                        Douglas Instruments
                                                          Per    Per    Per             Worth
                                                          year   week   day           Automating?              Assumptions

                                            Structures    70     1.4                                                 Target: structures per year   70
                                      Purified proteins          9.8     2                                   Purified proteins/structure (SPINE)   7

                                            Screening:
                                                 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    1
                                                 Plates          4.2    0.8                                              Plates per optimization   3
                                                                               YES, but it’s not easy (use
                         Reservoirs to be dispensed                      81          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    4
                                    Crystals to mount            17      3                NO                       Crystals per structure (UGA)    12



Microbatch seminar- slide 5
                          Automation: don’t over-automate!




                                                                            Douglas Instruments
               „          Recovery from errors can be very time-consuming

               „          Avoid long chains of automatic systems

               „          Use human buffer zones e.g. move plates by hand




Microbatch seminar- slide 6
                                                                               Douglas Instruments
          1. Degree of automation

          2. Crystallization methods (with phase diagrams)

          3. Experimental design ‟ steps of protein crystallization projects




Microbatch seminar- slide 7
                              Vapor diffusion




                                                Douglas Instruments
Microbatch seminar- slide 8
                               Phase diagram of a protein




                                                                                       Douglas Instruments
                                                                       precipitation

                                        nucleation

[Protein]


                                                     metastable zone

                                clear


                                              [Precipitant]
 Microbatch seminar- slide 9
                                                                                                       Douglas Instruments
               Thermodynamic processes which develop so slowly as to allow each intermediate step to
               be an equilibrium state are said to be reversible processes.




Microbatch seminar- slide 10
                                Phase diagram of a protein




                                                                               Douglas Instruments
                                                                        p
                                                    n


[Protein]

                                                                        m.z.

                                  Vapor diffusion




                                                        [Precipitant]
 Microbatch seminar- slide 11
                                    Vapor diffusion




                                                              Douglas Instruments
               „ Works well

               „ Gentle ‟ drop is concentrated AFTER mixing

               „ Doesn’t suit all proteins




Microbatch seminar- slide 12
                               Dialysis




                                          Douglas Instruments
Microbatch seminar- slide 13
                                Phase diagram of a protein




                                                                             Douglas Instruments
                                                                      p
                                                  n
                                Dialysis



[Protein]

                                                                      m.z.
                                           V.D.




                                                      [Precipitant]
 Microbatch seminar- slide 14
                                          Dialysis




                                                     Douglas Instruments
               „ Gives a lot of control

               „ You have to be patient

               „ Not easy to automate




Microbatch seminar- slide 15
                               Microbatch




                                            Douglas Instruments
Microbatch seminar- slide 16
                                Phase diagram of a protein




                                                                                       Douglas Instruments
                                                                                p
                                                  n
                                Dialysis



[Protein]                                                      M.B (paraffin)




                                                                                m.z.
                                           V.D.




                                                      [Precipitant]
 Microbatch seminar- slide 17
                                 Phase diagram of a protein




                                                                                                   Douglas Instruments
                                                                                            p
                                                              n
                                    Dialysis



[Protein]                                                                  M.B (paraffin)




                                                                                            m.z.
                                                       V.D.


                                M.B. (Si / paraffin)



                                                                  [Precipitant]
 Microbatch seminar- slide 18
                                Phase diagram of a protein




                                                                                                    Douglas Instruments
                                                                                             p
                                                          n
                                 Dialysis


                                                                      M.B (p) OPTIMIZATION
[Protein]

                                                                                             m.z.
                                                   V.D.


                                M.B. (Si/p) SCREENING



                                                              [Precipitant]
 Microbatch seminar- slide 19
                                       Microbatch




                                                                                       Douglas Instruments
         „ Simple and cheap

         „ Versatile ‟ screening / optimization, different oils, additives, volatile
           reagents (ethanol, iso-propanol etc.)

         „ Suits some proteins very well




Microbatch seminar- slide 20
                               Counter-diffusion




                                                   Douglas Instruments
Microbatch seminar- slide 21
                                Phase diagram of a protein




                                                                                                  Douglas Instruments
                                                                                           p
                                Counter-diffusion

                                                             n
                                  Dialysis



[Protein]                                                                 M.B (paraffin)




                                                                                           m.z.
                                                      V.D.


                                 M.B. (Si/paraffin)



                                                                 [Precipitant]
 Microbatch seminar- slide 22
                                            Counter-diffusion




                                                                                  Douglas Instruments
               „         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




Microbatch seminar- slide 23
                                            Counter-diffusion




                                                                                  Douglas Instruments
               „         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




Microbatch seminar- slide 24
       What % volume of protein should you use?




                                                   Douglas Instruments
                               100 nl + 100 nl ?

                               200 nl + 100 nl ?


                                 1 µl + 1 µl ?

                                 2 µl + 1 µl ?



Microbatch seminar- slide 25
                     What % of protein should you use?




                                                                                  Douglas Instruments
                               Microbatch with Si. / Par.:


                                   n
                  [Protein]


                                                             m.z.

                                                                    Precipitant
                                                                     saturated




                                        [Precipitant]

Microbatch seminar- slide 26
                     What % of protein should you use?




                                                                                                                  Douglas Instruments
                                               Microbatch with Si. / Par.:


                                                   n
                  [Protein]

                               Protein stock
                                                                                             m.z.

                                                          50%                                       Precipitant
                                                                                                     saturated


                                                                             Precipitant stock

                                                        [Precipitant]

Microbatch seminar- slide 27
                     What % of protein should you use?




                                                                                                                  Douglas Instruments
                                               Microbatch with Si. / Par.:


                                                   n
                  [Protein]

                               Protein stock
                                                                                             m.z.
                                                   66%
                                                          50%                                       Precipitant
                                                                                                     saturated


                                                                             Precipitant stock

                                                         [Precipitant]

Microbatch seminar- slide 28
       What % volume of protein should you use?




                                                                                                            Douglas Instruments
      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




Microbatch seminar- slide 29
                                                                               Douglas Instruments
          1. Degree of automation

          2. Crystallization methods (with phase diagrams)

          3. Experimental design ‟ steps of protein crystallization projects




Microbatch seminar- slide 30
                               Conventional Approach




                                                       Douglas Instruments
               1. Screening ‟ get first crystals




               2. Optimization ‟ improve crystals




Microbatch seminar- slide 31
                                     Experimental Design Steps




                                                                 Douglas Instruments
              Step 1. “Primary Screen.”
              Approx. 60-dimensional search.




              Step 2. “Targeted Screen”
              Approx. 12-dimensional search.




              Step 3. “Multidimensional Grid”
              Approx. 5-dimensional search.




Microbatch seminar- slide 32
                                      Experimental Design Steps




                                                                  Douglas Instruments
              Step 0. “Prescreen” to find precipitation points
              1-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.


Microbatch seminar- slide 33
                                  Experimental Design Steps
              Step 0. “Prescreen” to find precipitation points




                                                                 Douglas Instruments
              1-dimensional search.
               E.g. Pre-crystallization assay,
                       Pre Screening Assay,
                       Footprint Screen

              „ Use to adjust protein concentration
              „ Automation is available




Microbatch seminar- slide 34
                                 Experimental Design Steps




                                                             Douglas Instruments
             Step 1. “Primary Screen.”
             Approx. 60-dimensional search.
              E.g.        Sparse Matrix

             „ Many robotic systems are available
             „ Use pre-mixed solutions




Microbatch seminar- slide 35
                                 Experimental Design Steps




                                                             Douglas Instruments
             Step 2. “Targeted Screen”
             Approx. 12-dimensional search.
             1. Additive approach
             2. De novo approach




Microbatch seminar- slide 36
                                  Step 3: “Targeted Screen”




                                                                         Douglas Instruments
   1. Additive approach
   e.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:




Microbatch seminar- slide 37
                                          Step 3: “Targeted Screen”




                                                                                    Douglas Instruments
   1. Additive approach
   e.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 4.6
                     2.2       0.18M Mg formate + 0.1M Na citrate pH 6.5
                     2.3       0.18M Mg formate + 4% w/v PEG 8000
                     2.4       0.18M Mg formate + 4% w/v 2-methyl-2,4-pentanediol
                                ……………. etc.




Microbatch seminar- slide 38
                                          Step 3: “Targeted Screen”




                                                                                    Douglas Instruments
   1. Additive approach
   e.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 4.6
                     2.2       0.18M Mg formate + 0.1M Na citrate pH 6.5
                     2.3       0.18M Mg formate + 4% w/v PEG 8000
                     2.4       0.18M Mg formate + 4% w/v 2-methyl-2,4-pentanediol
                                ……………. etc.




Microbatch seminar- slide 39
                                          Step 3: “Targeted Screen”




                                                                                    Douglas Instruments
   1. Additive approach
   e.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 4.6
                     2.2       0.18M Mg formate + 0.1M Na citrate pH 6.5
                     2.3       0.18M Mg formate + 4% w/v PEG 8000
                     2.4       0.18M Mg formate + 4% w/v 2-methyl-2,4-pentanediol
                                ……………. etc.


   2. De novo approach
   e.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:




Microbatch seminar- slide 40
                                          Step 3: “Targeted Screen”




                                                                                    Douglas Instruments
   1. Additive approach
   e.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 4.6
                     2.2       0.18M Mg formate + 0.1M Na citrate pH 6.5
                     2.3       0.18M Mg formate + 4% w/v PEG 8000
                     2.4       0.18M Mg formate + 4% w/v 2-methyl-2,4-pentanediol
                                ……………. etc.


   2. De novo approach
   e.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 4000
                  3.3 25% w/v PEG 1500 + 0.1M Na acetate pH 4.6
                  3.4 20% w/v PEG 4000 + 4% w/v 2-methyl-2,4-pentanediol
                         ……………. etc.
Microbatch seminar- slide 41
                                          Step 3: “Targeted Screen”




                                                                                    Douglas Instruments
   1. Additive approach
   e.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 4.6
                     2.2       0.18M Mg formate + 0.1M Na citrate pH 6.5
                     2.3       0.18M Mg formate + 4% w/v PEG 8000
                     2.4       0.18M Mg formate + 4% w/v 2-methyl-2,4-pentanediol
                                ……………. etc.


   2. De novo approach
   e.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 4000
                  3.3 25% w/v PEG 1500 + 0.1M Na acetate pH 4.6
                  3.4 20% w/v PEG 4000 + 4% w/v 2-methyl-2,4-pentanediol
                         ……………. etc.
Microbatch seminar- slide 42
                               Step 3: “Targeted Screen”




                                                                                 Douglas Instruments
   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


Microbatch seminar- slide 43
                               Experimental Design Steps




                                                            Douglas Instruments
    Step 3. “Multidimensional Grid”
    Approx. 5-dimensional search.
    E.g. Central Composite, Box Behnken, XSTEP Autodesign




Microbatch seminar- slide 44
                               Multivariate experimental design




                                                                                                 Douglas Instruments
                    Almost all protein crystallization experiments have at least 4 parameters:
                                  1.    Protein concentration
                                  2.    Precipitant concentration
                                  3.    pH
                                  4.    Temperature
                                  5.    Additive ? …………….




Microbatch seminar- slide 45
                               Central Composite design




                                                          Douglas Instruments
Microbatch seminar- slide 46
                               Box-Behnken design




                                                    Douglas Instruments
Microbatch seminar- slide 47
                The Autodesign function of XSTEP ….




                                                      Douglas Instruments
Microbatch seminar- slide 48
            …. automatically fills a “spreadsheet” …




                                                       Douglas Instruments
Microbatch seminar- slide 49
                               …. and XSTEP executes it.




                                                           Douglas Instruments
Microbatch seminar- slide 50
                                    Experimental Design Steps




                                                                Douglas Instruments
                    Step 4. “2-D Grid”
                    Approx. 2-dimensional search.
                    E.g. XSTEP grids, manual experiments
Microbatch seminar- slide 51
                               Xstep Optimization 2-d grid




                                                             Douglas Instruments
Microbatch seminar- slide 52
                                         2-Dimensional grids




                                                                                                              Douglas Instruments
                               „   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




Microbatch seminar- slide 53
                                      Experimental Design Steps




                                                                  Douglas Instruments
              Step 0. “Prescreen” to find precipitation points
              1-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.


Microbatch seminar- slide 54
                                    Reducing the dimensions that must be considered
                                    60    60 dimensions (ingredients)




                                                                                      Douglas Instruments
Number of dimensions that you are




                                    24
         thinking about




                                    12




                                    5


                                    2



                                                                        TIME
      Microbatch seminar- slide 55
                                    Reducing the dimensions that must be considered
                                    60




                                                                                      Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
         thinking about




                                    12




                                    5

                                             ?
                                    2



                                                             TIME
      Microbatch seminar- slide 56
                                    Reducing the dimensions that must be considered
                                    60




                                                                                      Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
         thinking about




                                    12




                                                      Thinking ……
                                    5

                                             ?
                                    2



                                                                    TIME
      Microbatch seminar- slide 57
                                    Reducing the dimensions that must be considered
                                    60




                                                                                      Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
         thinking about




                                    12




                                    5

                                             ?
                                    2



                                                             TIME
      Microbatch seminar- slide 58
                                    Reducing the dimensions that must be considered
                                    60




                                                                                            Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
                                                                    Additve-type targeted
                                                                    screen
         thinking about




                                    12




                                    5

                                             ?                          ?
                                    2



                                                             TIME
      Microbatch seminar- slide 59
                                    Reducing the dimensions that must be considered
                                    60




                                                                                                           Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
                                                                                   Additve-type targeted
                                                                                   screen
         thinking about




                                    12


                                                             De novo targeted
                                                                       screen

                                    5

                                             ?                                         ?
                                    2



                                                                            TIME
      Microbatch seminar- slide 60
                                    Reducing the dimensions that must be considered
                                    60




                                                                                                                            Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
                                                                                   Additve-type targeted
                                                                                   screen
         thinking about




                                    12


                                                             De novo targeted
                                                                       screen
                                                                                                           Multi-d screen
                                    5

                                             ?                                         ?
                                    2

                                                                                                            ?
                                                                            TIME
      Microbatch seminar- slide 61
                                    Reducing the dimensions that must be considered
                                    60




                                                                                                                                           Douglas Instruments
                                            Primary screen
Number of dimensions that you are




                                    24
                                                                                   Additve-type targeted
                                                                                   screen
         thinking about




                                    12


                                                             De novo targeted
                                                                       screen
                                                                                                           Multi-d screen
                                    5

                                                                                                                            2-d grid
                                             ?                                         ?
                                    2

                                                                                                            ?                          ?
                                                                            TIME
      Microbatch seminar- slide 62
                                            Douglas Instruments
                               Finally ….




Microbatch seminar- slide 63
                                           Oryx (arabian)




                                                            Douglas Instruments
                       The Biblical Zoo in Jerusalem




Microbatch seminar- slide 64

								
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