1-888-780-8535 © 2008 1-888-780-8535 © 2009 www.emeraldbiosystems.com U.S. Patents #7,129,091, #6,409,832 and Patents Pending 1 The Microcapillary Protein.

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Presentation transcript:

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending 1 The Microcapillary Protein Crystallization System MPCS TM This work supported in part by NIGMS NCRR co-sponsored PSI-2 Grant U54 GM Cory Gerdts Enabling Technologies Workshop 3/4/09

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending MPCS Competitive Advantages: Low Volume (5-10 nL Protein / experiment) Diffraction-Ready TM Crystals – translation / scale up not required Crystals easily extracted from Peel-Apart TM CrystalCards No Dead Volume – Every nL Gets Used In situ diffraction ~22  L performs a gradient of 96 conditions 2

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Zheng B.; Roach L. S.; R. F. Ismagilov J. Am. Chem. Soc. 2003, 125: Plug-Based Protein Crystallization Zheng, B.; Ismagilov, R.F. Angew. Chem. 2005, 117: H. Song, J. D. Tice, R. F. Ismagilov Angew. Chem.-Int. Edit. 2003, 42, 768.

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending CrystalCard™ 20 nl Plugs 200  m microcapillary How Does it Work? 4

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending CrystalCard™ 20 nl Plugs 200  m microcapillary 5 How Does it Work?

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Efficient Optimization of Crystal Growth by Fine Gradient Screening pH 6pH 7 pH 8 Optimize crystal growth using fine control over chemical gradients for: Protein, Buffers (pH), Salts, Ligands, Additives, Precipitants, etc. 20 nl Plugs 7

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending A Smooth Chemical Gradient with the MPCS Single Crystal Precipitate Microcrystals Single Crystals Crystal Clusters Gerdts et al. Acta Cryst. (2008). D64, 1116–

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Bups 27 SSGCID and the MPCS Methionine-R-sulfoxide reductase of Burkholderia pseudomallei Bups a 1.7 Å PDB ID: 3CXK Bups b.A1 2.9 Å PDB ID: 3EOO Gerdts et al. Acta Cryst. (2008). D64, 1116–1122 Bups 69 Bups 35 Bups 75 Bups 33 + Met Bups 14 Bups 33 9

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Spacer Gas Bubble Spacer Protein Li, L., et al. (2006). PNAS. 103, Pre-formed cartridges of crystallization reagents are combined with chemical gradients for efficient screening. ~22  L performs a gradient of 96 conditions (up to 3500 individual experiments) Hybrid Method: Sparse Matrix + Fine Chemical Gradient Screening 11

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending No Dead Volume – Every nL is Used The Plug Maker aspirates protein into one of the tubes connected to a syringe that is back-filled with the carrier fluid (cf). Every nL of protein gets displaced into the CrystalCard. cf Syringe filled with carrier fluid 12

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending detector X-Rays MPCS™ CrystalCard™ (Side view) 30° In situ X-ray Diffraction Schematic goniometer head Beam Line Requirements: In-Line Optics for Crystal Aligment Stable and Accurate Motion Control Small Intense Beam Novel High Resolution Detectors 13

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending detector goniometer head X-Rays MPCS™ CrystalCard™ (Side view) 30° In situ X-ray Diffraction Schematic 100  m 1 mm Beam Line Requirements: In-Line Optics for Crystal Aligment Stable and Accurate Motion Control Small Intense Beam Novel High Resolution Detectors 14

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending In situ X-ray Diffraction Screening and Data Collection Crystals grown in the CrystalCard were subjected - in situ – to X- rays. Data collection at the NE-CAT beamline 24ID-C at APS Data merged from 3 crystals to generate a 1.9 Å resolution electron density map In situ Screening performed at GM/CA 23B CrystalCard goniometer holder being developed Gerdts et al. Acta Cryst. (2008). D64, 1116–

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending MPCS User Facility Visit MPCS User Facility ~5 miles from APS at ANL 2501 Davey Road, Woodridge, IL Development of the NIGMS NCRR co-sponsored PSI-2 Grant U54 GM MPCS-Beta Instrument

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending The Microcapillary Protein Crystallization System (MPCS™) Plug Maker™ & CrystalCards™ PSI Appreciation Discount: 30% Contact us TODAY for a Quotation: In appreciation of the ATCG3D and PSI funding, we are introducing the MPCS with a 30% discount! Discount offered for limited time only. Contact us for details.U.S. Patents #7,129,091, #6,409,832 and Patents Pending

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Touch-Screen User Interface Small Footprint (~18”x15”x14”) No Dead Volume Low Volume (5-10 nL Protein / Plug) ~22  L Performs Gradient of 96 Conditions Diffraction-Ready Crystals – “Translation” not Required Automated Crystallization Screening – Sparse Matrix and Gradient Optimization Fully Integrated Experimental Setup (Hardware/Software) The Plug Maker™ 18

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Automated Sampling 19

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Loaded and Ready 20

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending MPCS Competitive Advantages: “I can’t get my crystals out” 21 MPCS: Other Microfluidic Crystallization System(s): Peel-Apart TM CrystalCards TM “Crystal hits don’t translate or scale-up to vapor diffusion” Diffraction-Ready TM crystals – translation/scale-up unnecessary “Microchips are expensive” (minimum ~$0.63 per drop) More than 3X cheaper (~$0.19 per drop)

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending Videos - e-presentation (youtube) - Slides - User Facility

© © U.S. Patents #7,129,091, #6,409,832 and Patents Pending