Protein Crystals for Neutron Crystallography (PC4NC) Large volume crystal growth of Inorganic Pyrophosphatase complexes by counter-diffusion in microgravity for neutron diffraction studies (PC4NC) - 05.13.15

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Enzymes play a critical role in many biological processes, but the way they operate is not well understood. Protein Crystals for Neutron Crystallography (PC4NC) studies an enzyme called inorganic pyrophosphatase (IPPase) to determine how it functions. By studying the diffraction of neutrons in the crystal, researchers can locate the positions of hydrogen bonds in the enzyme, which will help determine how the molecule works in a cell. In order to do so, large enzyme crystals must be grown, but this is only possible in the microgravity environment of the International Space Station.
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The following content was provided by Joseph D. Ng, Ph.D., and is maintained in a database by the ISS Program Science Office.
Experiment Details


Principal Investigator(s)
Joseph D. Ng, Ph.D., iXpressGenes, Hunstville, AL, United States

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Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Earth Benefits, Scientific Discovery

ISS Expedition Duration
March 2014 - September 2014

Expeditions Assigned

Previous ISS Missions
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Experiment Description

Research Overview

  • Complexes of the inorganic pyrophosphatase (IPPase) from the archaeon Thermococcus thioreducens are the targets for large volume crystal (1mm3) growth for Neutron Macromolecular Crystallography (NMC). Counter- diffusion crystallization in capillaries having diameters of 1mm or greater are used in the Granada Box Facility to achieve this goal. A microgravity environment is essential to form a stable supersaturation gradient to obtain these large crystals.  The principal outcome is to identify the role of H atoms in enzymatic activity and decipher a structure-based mechanism for IPPase and its complexes where this would not be possible otherwise with previous crystallographic models determined from Earth-grown crystals. 

PC4NC is supported by a technical and scientific hypothesis. The technical hypothesis is growing IPPase crystals by counter-diffusion equilibration in a large-diameter restricted cylindrical geometry under microgravity would provide large volume protein crystals that would be superior to those equivalent crystals grown on Earth or otherwise not possible to obtain.


The specific scientific hypothesis is the hydrolysis of inorganic pyrophosphate catalyzed by IPPase proceeds via direct phosphoryl transfer to water without the formation of a phophorylated enzyme intermediate.The mechanism is proposed to proceed by a nucleophilic attack by a water-metal activation. It is believed that an extended network of hydrogen bonding interactions mediate and perhaps dictate the architecture of the catalytic site affecting the degree of catalysis and substrate recognition of IPPase.Obtaining large volume crystals of all the IPPase complexes will reveal by NMC the true role of the structured solvent and dissociable hydrogen atoms. As a result, the location of hydrogen atoms and water molecules can be identified to support mechanistic studies that otherwise would not be possible.Researchers will address the aforementioned proposition with the following objectives.

  • To grow protein crystals in large-diameter capillaries in a diffusion limiting mass transport process on Earth and under microgravity.
    • IPPase complexes- over 25 different protein form IPPases is prepared to be crystallized by counter-diffusion in restricted geometry. The crystal growth process is observed and measured to further understand its equilibration process in different configurations.
    • D2O uptake by the crystal in the restricted geometry format is measured to quantify deuterium exchange rates and optimize D2O exchange protocols.
    • Perdeuterated IPPase is also crystallized in the same format to observe crystal growth differences due to isotope effects.
  • To determine the total protein structures of IPPase and other hyperthermophilic proteins by Neutron Crystallography.
    • Initial neutron diffraction analysis is performed on single large protein crystals for preliminary evaluation.
    • Complete neutron crystallographic data collection for selected proteins grown on Earth and those that have been obtained under microgravity are performed.
    • Neutron crystallographic data sets are coupled to already existing X-ray crystallographic structures for total protein structure determination. Since neutron diffraction does not cause any radiation damage, protein crystals that have been analyzed by NMC are also evaluated by synchrotron X-ray.
  • To obtain mechanistic models of protein catalysis and binding for selected proteins.
    • Determine hydrogen positions and protonation states of IPPases complexed to different metals, pyrophosphate substrates and inhibitors from neutron and X-ray diffraction data.
    • Biochemical characterization is performed involving kinetic and binding measurements to accompany structural data.
    • Propose a comprehensive molecular model of catalysis.


The investigation relies on the usage of the Granada Crystallization Facility (GCF). GCF is a crystallization encasement that allows counter-diffusion crystallization experiments in space using the Granada Crystallization Box (GCB) designed by the Laboratorio de Estudios Cristalograficos; (LEC) 20 and supported by the European Space Agency. The GCB has already been used in different missions in microgravity conditions: Andromede and Odissea for ESA and several missions with JAXA.


The GCB works as schematically shown in Fig. 1A,B. There is a gel buffer layer located at the bottom of the reservoir after inserting the guide. Once the gel is set, the capillaries are filled with the protein and inserted through the hole of the guide and punctuated to a given depth into the gelled buffer layer. Finally the precipitating solution is set on top of the buffer gel and the capillary tubes are sealed (usually with wax or enamel).During the equilibration process, the precipitant slowly diffuses through the gel interface against the protein chamber along the length of the capillary. A counter-diffusion scenario is set-up and the equilibration process proceeds as described previously in this proposal.The technique works by the coupling between diffusion mass transport and precipitation. Therefore counter-diffusion experiments must be implemented in an environment where the role of convection is reduced to be negligible. Thus, microgravity is a natural scenario to use it. Thus the GCB is designed to be used in space as a passive crystallization apparatus requiring neither crew time nor energy supply for activation/deactivation. Each of the boxes accommodates six capillaries with a maximum inner diameter of 1.5 mm. The external dimensions of the GCB are 33 x 100 x 7 mm. The weight of one GCB filled with six capillaries and the chemical solutions is 22 grams. GCB can be piled and therefore a large number of individual experiments can be performed using a small total volume and minimizing the total weight. These features thereby enable the meaningful statistical data required with different macromolecules.

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Space Applications
Unlike X-ray diffraction, which can also unveil the structure of a material, neutron diffraction requires large volumes of crystals, which are difficult to grow on Earth. Microgravity is an ideal environment for crystallizing large molecules like proteins, because gravity does not affect how the crystal structures form. More than 80,000 protein structures have been derived using X-ray diffraction, while only about 50 have been studied with neutron diffraction. The International Space Station may be an essential environment to obtain protein crystals suitable for neutron diffraction studies.

Earth Applications
Successfully determining the structure of IPPase would be a major breakthrough for medical science on Earth. Crystallizing the enzyme would reveal its mechanisms for the first time, allowing researchers to understand the atomic reactions involved in this type of transfer enzyme. Understanding how this enzyme works could enable the design of new antibiotics. It could also improve processing for a common type of DNA sequencing technology known as polymerase chain reaction, projected to be worth $27 billion by 2015.

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Operational Requirements
Experiment is completely passive and requires no resources except volume on the ISS.

Operational Protocols
No on-orbit operations except to transfer the payload from Dragon capsule to ISS for increment and from ISS to Dragon Capsule for return after increment.

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Results/More Information

Information Pending

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Related Websites

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Figure 1.  Granada crystallization box(GCB) and facility (GCF).  Counter diffusion crystallization can be performed in capillary filled with protein solution set to equilibrate with a precipitant that slowly diffuses across a gel. 

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