psi-2 bottlenecks meeting, april 14-16, 2008 a semi-automated system for nanovolume plug-based...
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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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Outline
Technology Background
Project Update
Upcoming Improvements
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Labcard
The Microfluidic Protein Crystallization System: (MPCS)
Slide prepared by Cory Gerdts
A Three Component System
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Crystallization in Plugs
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Hybrid Method
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Outline
Technology Background
Project Update
Upcoming Improvements
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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)
Slide prepared by Cory Gerdts
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
MPCS - Beta
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Multiple-Pronged Approach: 2 Functional Labcards
Slide prepared by Cory Gerdts
SBS Formatted Plate
PSI-2 Bottlenecks Meeting, April 14-16, 2008
MPCS Advantages/Features: Diffraction-Ready Crystals
Slide prepared by Cory Gerdts
Reaction Center
Fatty Acid Amide Hydrolase
Acyl-CoA hydrolase
Porin
Myoglobin
TDP
ribose-phosphate pyrophosphokinasemethionine-R-
sulfoxide reductase
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Protein Crystal Extraction
Slide prepared by Cory Gerdts
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Improve efficiency:
*Mutants * Ligands *DNA complexes
pH 6 pH 7 pH 8
* Cryoprotectants * Ligands * Other Protein Partners * Other DNA Partners * Detergents * Heavy Atoms
Slide prepared by Cory Gerdts
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Results of a Smooth Gradient
PSI-2 Bottlenecks Meeting, April 14-16, 2008
• 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.
Slide prepared by Cory Gerdts
MPCS Advantages/Features: No Dead Volume
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Cory Gerdts
MPCS Advantages/Features: Microfluidic Seeding
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Outline
Technology Background
Project Update
Upcoming Improvements
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Microcapillary Crystallization: Next Steps
Development of more user friendly full instrument
Improved connections (hybrid in plastic Labcards)
Automatic Aspirator
Improved pumps
Further Dissemination
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Acknowledgements
Microcapillary Crystallization
deCODE: 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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Space group – P1 (Triclinic)
Unit cell – a = 42.00, b = 45.17, c = 45.40
Angles – = 88.4, = 83.7, = 69.1
Scaled 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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Lance Stewart
PSI-2 Bottlenecks Meeting, April 14-16, 2008
Simple 4-Microsyringe Pump System and “Microplugger” Control Software and Labcard Prototype
pH Gradient Test, ~10nl Plugs
Slide prepared by Lance Stewart
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Lance Stewart
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Lance Stewart
3D in 3 Day Challenge
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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)
Slide prepared by Lance Stewart
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Cory Gerdts
www.MPCW2008.org
PSI-2 Bottlenecks Meeting, April 14-16, 2008
PSI-2 Bottlenecks Meeting, April 14-16, 2008
• Storage capillary controls evaporation
• Membrane protein crystallization is done using Teflon capillaries and PDMS 3+1 mixers.
Slide prepared by Cory Gerdts
MPCS Advantages/Features: Membrane Proteins
PSI-2 Bottlenecks Meeting, April 14-16, 2008
ATCG3D
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
Slide prepared by Cory Gerdts
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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
PSI-2 Bottlenecks Meeting, April 14-16, 2008
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