A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION

PSI-2 Bottlenecks Meeting, April 14-16, 2008

A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION

Cory Gerdts, Ph.D.April 15, 2008

PSI-2 Bottlenecks Meeting, Natcher Conference Center April 14-16, 2008

ATCG3D is a Specialized PSI-2 Center Funded by the NIGMS and NCRR, Grant # U54-GM074961-03

Slide prepared by Lance Stewart


Outline

Technology Background

Project Update

Upcoming Improvements

Slide prepared by Cory Gerdts


H. Song, J. D. Tice, R. F. Ismagilov Angew. Chem.-Int. Edit. 2003, 42, 768

Langmuir (2003)Appl. Phys. Lett. (2003) Anal. Chim. Acta (2004)

Philos. T. Roy. Soc. A (2004)

fluorocarbon

Droplets (Plugs) in Microfluidic Channels



Labcard

The Microfluidic Protein Crystallization System: (MPCS)


A Three Component System


Crystallization in Plugs



Formation of Fine Gradients

Zheng B.; Roach L. S.; R. F. Ismagilov J. Am. Chem. Soc. 2003, 125: 11170-11171.

Zheng, B.; Ismagilov, R.F. Angew. Chem. 2005, 117: 2576-2579.

Pre-formed Cartridge - ~10 nL plugs

200 m

Gradient and Sparse Matrix Screening

Sparse Matrix Screening with Pre-Formed Cartridges



Hybrid Method: Sparse Matrix + Gradient Screening

+

L. Li, D. Mustafi, Q. Fu, V. Tereshko, D.L. Chen, J.D. Tice, R.F. Ismagilov PNAS, 2006, 103, 19243.Slide prepared by Cory Gerdts


Hybrid Method



Outline


Project Update




MPCS Development Goals

Labcard Flat Thin Rigid Low Dead Volume Macro-

Micro interface Injection Molded Parts (low

cost) One-time Use Plastic Material Properties:

TransparentLow BirefringenceX-ray TransmissiveLow Surface Energy

(hydrophobic/fluorophilic)


Pump System Constant Flow at Low Flow

Rates Ability to Form Smooth

Gradients No Delay/Response Time

Dissemination Disseminate Technology to

Center and Community Make Technology Available


MPCS - Beta


Multiple-Pronged Approach: 2 Functional Labcards


SBS Formatted Plate


MPCS Advantages/Features: Diffraction-Ready Crystals


Reaction Center

Fatty Acid Amide Hydrolase

Acyl-CoA hydrolase

Porin

Myoglobin

TDP

ribose-phosphate pyrophosphokinasemethionine-R-

sulfoxide reductase


Protein Crystal Extraction


Methionine-R-sulfoxide Reductase data set: 1.7 Å resolution

PSI-2 Bottlenecks Meeting, April 14-16, 2008Slide prepared by Cory Gerdts

MPCS Advantages/Features: In Situ Diffraction

Data merged from 3 crystals: 1.9 Å resolution


Improve efficiency:

*Mutants * Ligands *DNA complexes

pH 6 pH 7 pH 8

* Cryoprotectants * Ligands * Other Protein Partners * Other DNA Partners * Detergents * Heavy Atoms


MPCS Advantages/Features: Optimization

Applications of small volume fine gradient screening: Optimize buffer system, precipitant concentration, and pH

Identify optimal or tolerable doses of additives to known crystallization conditions

Reduce protein preparatiion requirements for screening


Results of a Smooth Gradient


• 4 L of protein is enough for ~800 experiments. As little as 0.5 L protein can be aspirated easily.

• Protein is aspirate into a piece of Teflon tubing on a syringe that is back-filled with the fluorocarbon (FC) oil. Every nL of protein gets displaced into the system.


MPCS Advantages/Features: No Dead Volume


3. The Labcard is filled with plugs that each contain one or a few small microcrystals.

Microcrystals from a previous trial can be used to performmicrofluidic seeding in the MPCS

1. Microcrystals are aspirated into a Teflon tube

2. The microcrystal-containing solution is used as one of the aqueous

streams in the 3+1 mixer.

Microfluidic seeding can be used to:• Optimize a protein that nucleates too much, or

• Induce crystallization in a mutant, ligand, DNA, etc. screen by crushing up a crystal from a known condition or from the wild type version


MPCS Advantages/Features: Microfluidic Seeding


Outline


Project Update




Microcapillary Crystallization: Next Steps

Development of more user friendly full instrument

Improved connections (hybrid in plastic Labcards)

Automatic Aspirator

Improved pumps

Further Dissemination


Microcapillary Protein Crystallization Workshop

Date: June 6-7, 2008

Location: deCODE biostructuresEmerald BioSystems

2501 Davey Road Woodridge, IL 60517

Micropipet / MicrocapillaryHandling and Crystallization

Counterdiffusion Microcapillary Crystallization

Plug-Based Microcapillary Crystallization

Slide prepared by Cory Gerdts and Lance Stewart


Acknowledgements

Microcapillary CrystallizationdeCODE: Lance Stewart (PI) Peter Nollert Mark Elliot Yiping Xia

The Scripps Research Institute: Raymond Stevens (Co-PI) Peter Kuhn Peter Clark Vadim Cherezov Joe Ng

ATCG3D Funding NIGMS / NCRR U54-GM074961

University of Chicago: Rustem Ismagilov Liang Li George Sawicki Debarshi Mustafi Valentina Terechko Qiang Fu Wenbin Du Anna Selezneva

For more information:Cory Gerdts

Phone: 630-783-4691Email: cgerdts@

emeraldbiosystems.com


Space group – P1 (Triclinic)Unit cell – a = 42.00, b = 45.17, c = 45.40

Angles – = 88.4, = 83.7, = 69.1Scaled from 50 – 1.7

Completeness – 95.3 (87.4)Rmerge – 6.8% (42.3)

I/I – 23.1 (2.2)# of reflections -118,181 (total), 32,539 (unique)

Redundancy 3.6 (3.3)

Methionine-R-sulfoxide Reductase data set


Counterdiffusion Confined Geometry Microcapillary Crystallization

Topas COC plastic microfluidic crystallization Greiner Bio-One labcard

Individual Lysozyme Crystals grown in microcapillary channels by counterdiffision methods

In situ X-ray diffraction on individual crystals reveals different diffraction quality

Slide prepared by Lance Stewart from data of Joe Ng and Peter Kuhn

Greiner Bio-One Commercialization of Confined Geometry Counterdiffusion Labcard

PrototypePrototype

Prototype Design


USABLE soluble AND membrane protein crystals grow in ~10 nL plugs In situ diffraction for both data acquisition and crystal quality screening Increased efficiency from preparing decreasing amounts of protein On-chip formulation for fine granularity screening for “narrow crystallization slots” ~800 experiments per card No dead volume – every nL gets used Large Phase Space can be investigated with the Hybrid Method using preformed cartridges Microfluidic seeding

Summary of MPCS Advantages and Features



Nanovolume Microcapillary Crystallization with In Situ X-ray Imaging

Emerging Technologies Microfluidic Device Production (PDMS, Plastic, Glass) Microfluidic Pumping Systems (Various) Novel Microfluidic Circuitry and Methods for Protein Crystallization

(Seeding, Gradient, Random Sparse, Microbatch, Counterdiffusion)

Opportunity Efficient Exploration of Crystallization Space per Unit Protein Integration of Crystallization with X-ray Data Collection (In Situ)

Efficiency Gain Increased Crystallization Success per Unit of Protein Improved In situ Diffraction Quality Screening (true measure of a crystal) Improved Inventory Staging of Crystals for X-ray Data Collection



Simple 4-Microsyringe Pump System and “Microplugger” Control Software and Labcard Prototype

pH Gradient Test, ~10nl Plugs


Emerald BioSystems is offering commercial systems as of July 2007

In Situ X-ray Diffraction ofProtein Crystals in LabcardsLabcard PrototypeMicro-Syringe Pump System

Microplugger™ Control Software


ATCG3D Technology Focus Areas

Compact Light Source for Tunable X-ray Diffraction Data Collection in a Laboratory Setting

Nanovolume Microcapillary Crystallization with In Situ X-ray Imaging

Computer Aided Protein Construct Engineering and Synthetic Gene Design



DETECT-X Imaging of Protein Crystals in Microfluidic Devices

DETECT-X Imager used in conjunction with Millipol software

High signal to noise imaging of crystals

Crystal orientation imaging

Potential to Enable Efficient Complete in situ X-ray diffraction Data Set Collection

Slide prepared by Lance Stewart with data from Peter Nollert

W. Kaminski, Millipol SoftwareNorgren Systems LLC Hardware Engineering

Emerald BioSystems, Commercial Launch of DETECT-X, Q2 2007


Future Directions Gene-to-3D in 3 Days ?

Starting from Oligonucleotides

PCR based gene production / transcription mRNA, 8 h Cell free translation for protein production, 16 h Affinity purification of protein, 4 h Nanovolume microcapillary labcard crystallization, 20 h In situ X-ray diffraction data collection, 6 h Structure Determination 16 h

TOTAL ~70 h


3D in 3 Day Challenge


Acknowledgements

Synthetic Gene Design Software John Walchli (deCODE) Alex Burgin (deCODE) Mark Mixon (deCODE) Don Lorimer (deCODE)

ATCG3D PI Collaborators Peter Kuhn and Raymond Stevens

(Co-PIs, The Scripps Research Institute)


Corporate Collaborators Peter Nollert, Emerald BioSystems Lawrence Kuo, J&JPRD Masaki Madono, Cell Free Sciences

PSI-2 Grant Funding ATCG3D Funding NIGMS / NCRR

U54-GM074961

PSI-2 / SG Collaborators James Love, NYCOMPS Dmitriy Vinarov, CESG, Univ. of Wisconsin Mark Sullivan, University of Rochester Alexei Brooun, TSRI now at Pfizer Yaeta Endo, Ehime University

Synthetic Gene Design Wet Lab Work Don Lorimer (deCODE) Ellen Wallace (deCODE) Amy Raymond (deCODE) Adrienne Metz (deCODE) Rena Grice (deCODE)

SSGCID (NIAID) Collaborators Peter Myler, Seattle Biomedical Research Institute Wes Van Voorhis, University of Washington

Contract Funding SSGCID Funding from NIAID

HHSN272200700057C


Microcapillary Protein Crystallization Workshop

Date: June 6-7, 2008

Location: deCODE biostructures/Emerald BioSystems

2501 Davey Road Woodridge, IL 60517

Micropipet / MicrocapillaryHandling and Crystallization

Counterdiffusion Microcapillary Crystallization

Plug-Based Microcapillary Crystallization


www.MPCW2008.org


• Storage capillary controls evaporation

• Membrane protein crystallization is done using Teflon capillaries and PDMS 3+1 mixers.


MPCS Advantages/Features: Membrane Proteins


ATCG3D


Becoming a Beta User of theMicrocapillary Protein Crystallization System

Beta User obtains complete Microcapillary Protein Crystallization System

Training: #1: training of Beta User scientists at Emerald site (Bainbridge Island, WA)

Installation: complete installation of the Microcapillary Protein Crystallization System in Beta User laboratory, validation and QC

Training #2: training of Beta User scientists at Beta User site Cost: $24k for system, $19k for annual technology access license Emerald solicits feedback to improve system performance Beta User agrees not to reverse engineer system, resulting IP owned by

Emerald, co-authorship of publication


Nanovolume Protein Crystallization: PDMS 3+1 Mixer and Microcapillary Holder

PDMS 3+1 Mixer

0.45 meter of Teflon capillary stored in glass capillary

1 meter of tubing, ~1000 membrane

protein crystalliization

trials

Li, L., Mustafi, D., Fu, Q., Tereshko, V., Chen, D.L., Tice, J.D., Ismagilov, R.F. PNAS 2006, 103: 19243



Crystallization of Oligoendopeptidase Fby Microfluidic Seeding

Microfluidic Separation of Nucleation andCrystal Growth Yields Single Crystals

Target: apc36224 selected by MCSG

but unsolved

Only Precipitation and Crystal Clusters inOptimized Vapor Diffusion Experiments


Oligoendopeptidase F(B. stearothermophilus) Structure

Oligoendopeptidase F apc36224 3.1 Å resolution Space group: P3121; Unit Cell Parameters a=b 119.50 c=248.90 R-factor = 0.196, R-free = 0.248 Solvent Content: ~70%; PDB Accession #s 2H1N and 2H1J

Zn++ Metallopeptidase family M3 Structural similarity to:

human testicular Angiotensin-converting enzyme, 1O86

E.coli Dipeptidyl Carboxypeptidase Dcp, 1Y79 Neurolysin, 1I1I Pyrococcus furiosus carboxypeptidase, 1K9X

ATCG3D and MCSG collaboration Gerdts, C.J., Tereshko, V., Yadav, M.K., Dementieva, I., Collart, F., Joachimiak, A., Stevens, R.C., Kuhn, P.,

Kossiakoff, A., and Ismagilov, R.F. Angew. Chem. Int. Ed. 2006 45: 8156-8160

Time-Controlled Microfluidic Seeding in nL-Volume Droplets To Separate Nucleation and Growth Stages of Protein Crystallization

A SEMI-AUTOMATED SYSTEM FOR NANOVOLUME PLUG-BASED CRYSTALLIZATION

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