A Low Volume, High Density Microgravity Protein Crystal Growth Method Using a

NanoRacks NanoLab Carl W. Carruthers, Jr., HMRI; Cory Gerdts, Protein BioSolutions;

Michael D. Johnson, NanoRacks, LLC; Paul Webb, HMRI

CCarruthers@nanoracks.com @proteinwrangler

Outline

• Rationale for Microgravity Protein Crystal Growth (mg PCG)

• Goal and Overview of Methods for This Project

• Results of this Project

Why Study Protein?

• “Everything comes down to protein interactions.”

• X-ray crystallography is a powerful technique to study, at an atomic level, the 3D structure a protein.

• Structures lead to deep understanding of mechanisms of life and possible therapeutics for diseases.

-Dr. Nick Pace, TAMU

Protein Structure Determination

“Crystallography: Atomic secrets. 100 years of crystallography.” Nature 29 January 2014

Photos: Wikipedia

Diagram: Cornell

Protein Structure Determination

1g Convection/Sedimentation Can Create Irregular Crystals

McPherson, A., Malkin, A.J., Kuznetsov, Y.G., Koszelak, S., Wells, M., Jenkins, G. Howard, J. Lawson, G. "The effects of microgravity on protein crystallization: evidence for concentration gradients around crystals." Journal of Crystal Growth. 196 (1999) 572-586.

Optimal Spot Spot Overlap Spot Smearing

Microgravity Protein Crystal Growth

• ~270 proteins have flown on Space Shuttle Program in 25 years

• Experiments were for improving existing models or studying how crystals grow

• Technology to grow crystals reached significant level of complexity

Device PCG Type # of Expts

Vapor diffusion apparatus-2 (VDA-2) Vapor diffusion 20

Commercial vapor diffusion apparatus,(CVDA) Vapor diffusion 128

Protein crystallization facility (PCF) Temperature Controlled ?

Dynamically Controlled Protein Crystal Growth System (DCPCG)

Vapor diffusion & Temperature Controlled

?

High Density Protein Crystal Growth System (HDPCG)

Vapor diffusion 1008

Hand Held Protein Crystallization Apparatus for Microgravity (HH-PCAM)

Vapor diffusion

24

Protein Crystallization Apparatus for Microgravity (PCAM)

Vapor diffusion

504

Diffusion-controlled Crystallization Apparatus for Microgravity (DCAM)

dialysis/liquid-liquid diffusion 81

Protein Crystal Growth-Enhanced Gaseous Nitrogen Dewar (PCG-EGN)

Batch/capillary ~500

Advanced Protein Crystallization Facility (APCF) hanging drop, FID, dialysis 48

Protein Crystallization Diagnostics Facility (PCDF) dialysis, batch 12

Gel acupuncture method (Grenada) Counter-diffusion capillary 138

Un

iver

sity

of

Ala

bam

a in

Hu

nts

ville

/NA

SA

ESA

JA

XA

/ESA

Snell, E. H., Helliwell, J.R. "Macromolecular crystallization in microgravity." Rep. Prog. Phys. 68 (2005) 799-853.

-Respiratory syncytial virus (RSV)

-Hepatitis C drug, Schering-Plough -JAXA: Duchenne's Muscular Dystrophy, Lipocalin - type Prostaglandin D Synthase 2 (L-PGDS) and Hematopoietic Prostaglandin D Synthase 2 (H-PGDS).

Outline

• Rationale for Microgravity Protein Crystal Growth (ug PCG)

• Goal and Overview of Methods for This Project

• Results of this Project

Device PCG Type # of Expts

Vapor diffusion apparatus-2 (VDA-2) Vapor diffusion 20

Commercial vapor diffusion apparatus,(CVDA) Vapor diffusion 128

Protein crystallization facility (PCF) Temperature Controlled ?

Dynamically Controlled Protein Crystal Growth System (DCPCG)

Vapor diffusion & Temperature Controlled

38-50

High Density Protein Crystal Growth System (HDPCG)

Vapor diffusion 1008

Hand Held Protein Crystallization Apparatus for Microgravity (HH-PCAM)

Vapor diffusion

24

Protein Crystallization Apparatus for Microgravity (PCAM)

Vapor diffusion

504

Diffusion-controlled Crystallization Apparatus for Microgravity (DCAM)

dialysis/liquid-liquid diffusion 81

Protein Crystal Growth-Enhanced Gaseous Nitrogen Dewar (PCG-EGN)

Batch/capillary ~500

Advanced Protein Crystallization Facility (APCF) hanging drop, FID, dialysis 48

Protein Crystallization Diagnostics Facility (PCDF) dialysis, batch 12

Gel acupuncture method (Grenada) Counter-diffusion capillary 138

Un

iver

sity

of

Ala

bam

a at

Bir

min

gham

/NA

SA

ESA

JA

XA

/ESA

Houston, We Have a Protein Crystal Growth Problem

• Despite advances in PCG ground hardware, mg hardware being used is lagging.

• Hardware uncommon to crystallographers

• Large sample volumes, low sample density

• Is there a COTS alternative for mg PCG equipment that’s low volume, high sample density?

Simple, Low Volume, High Density Experiment

Photo: Emerald Bio

- ~4 ml protein - 400-800 different conditions - Vary protein, buffer, and/or

precipitant concentration

Workflow

Diagram: NanoRacks

Experiment Goal & Set-up • Primary Goal: Test the ability to grow protein crystals in microgravity by

using this system. – 2nd Goal: Return of crystals

• 5 Proteins:

– Nuclear Receptor: • PPARg LBD

– Model Proteins

• Chicken Egg White Lysozyme • Thermolysin • Xylanase • Lipase B

• 3-5 different conditions (vary buffer, ppt & protein) each protein. • 25 flight & 25 ground controls

– Ground controls placed in identical NanoRacks NanoLab • In -80C till flight samples delivered to ISS, then in incubator at 24C

• Passive temperature control

Flight Samples & Ground Controls

Outline

• Rationale for Microgravity Protein Crystal Growth (ug PCG)

• Goal and Overview of Methods for This Project

• Results of this Project

Results

• Launched on SpaceX Falcon 9/Dragon March 1st/Delivered to ISS March 4th

• 1st on orbit survey: April 29, 2013

• 2nd on orbit survey of one card: May 8th, 2013

• Samples returned via Soyuz: May 14, 2013

1st On Orbit Survey: April 29, 2013

2nd On Orbit Survey: May 8, 2013

Sample Return: May 14, 2013

Sample Results

• 16 of 22 microgravity cards had crystals – PPARg LBD: 5 of 7 – Lysozyme: all 6 – Thermolysin: all 4 – Xylanase: 1 of 5

• 14 of 22 ground control cards had crystals – PPARg LBD: 2 of 7 – Lysozyme: all 6 – Thermolysin: all 4 – Xylanase: None

PPARg LBD

mg

1g

Lysozyme

mg

1g Red bar = 200mm

Thermolysin

mg

1g Red bar = 200mm

Xylanase

mg

1g

Red bar = 200mm

None

Experiment Results

• Primary Goal: Test the ability of growing protein crystals in microgravity by using this system.

– We only know for certain that one card had crystals that grew on orbit.

• Secondary Goal: Return of crystals

– 28h after landing and despite many temperature changes, lots of crystals still in slides.

Advantages of This System

• By combining Protein BioSolutions’ Plugmaker, CrystalCards™ and NanoRacks’ NanoLab, this method creates: – the ability of using a small quantity of protein to evaluate

hundreds of crystal growth conditions with each experiment having a footprint of a standard microscope slide.

– freezing of the filled CrystalCards™ makes it possible to

store them at -80C indefinitely, increases experiment flexibility with uncertain launch dates.

– retrieval of crystals is easily done by removing card seal or

if the investigator desires, the x-ray diffraction data can be collected while the crystal stays in the card.

Future Flights/System Modifications

• Next flight: CRS-4 August 2014

• Future Modifications:

– Active temperature control (4 or 22C)

– Automated microscope system

Thank You!

• Rolando Branly

• NanoRacks

• Jan-Ake Gustafsson

• Paul Webb