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ProMISS Protein Microscope for the I nternational Space Station

Dr. Ingrid Zegers 1 , Dr. Frank Dubois 2 1 Vrije Universiteit van Brussel Department of Ultrastructuur, Vlaams Interuniversitair Instituut Biotechnologie Paardenstraat 65, B-1640 St. Genesius Rode, Belgium Igzegers@vub.ac.be , Phone: +32 2 359 02 15, Fax: +32 2 359 02 89

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ProMISS Protein Microscope for the I nternational Space Station

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  1. Dr. Ingrid Zegers1, Dr. Frank Dubois2 1Vrije Universiteit van Brussel Department of Ultrastructuur, Vlaams Interuniversitair Instituut Biotechnologie Paardenstraat 65, B-1640 St. Genesius Rode, Belgium Igzegers@vub.ac.be, Phone: +32 2 359 02 15, Fax: +32 2 359 02 89 2Université Libre de Bruxelles Microgravity Research Center CP165-62, 50 Av. F. Roosevelt, 1050 Bruxelles Frdubois@ulb.ac.be, Phone: +32 2 650 42 83, Fax: +32 2 650 31 26 ProMISSProtein Microscope for the International Space Station

  2. Participants Scientific Background Proposed Experiment Experiment Hardware Experiment Scenario Summary Outline / Contents

  3. Participants GCB Granada Crystallisation box JuanMa Garcia-Ruiz (LEC, Granada) Optics Interferometry Jean-Claude Legros Frank Dubois ULB-MRC Modelling and similation Jean-Pierre Boon (ULB) Anne De Wit (ULB) Luigi Carotenuto (MARS, Naples) Protein crystallisation APCF Lode Wyns/Ingrid Zegers (VUB) Jean-Paul Declercq (UCL) Joseph Martial (Ulg) JuanMa Garcia-Ruiz (LEC) Adriana Zagari (Una) Experiment counterdiffusioncrystallisation with digital holography optics Develoment/construction ULB Pedeo Verhaert Astrium Busoc Marie-Claude Limbourg Emiel van Ransbeeck Etienne Haumont ESA Olivier Minster Vladimir Pletser other

  4. Why Protein crystallization? genome  protein - structure  function determining the structures of proteins: A Crystallization the diffraction pattern of a single molecule is too weak - with crystals the diffraction patterns of the well ordered molecules are ‘added up’ to each other. B X-ray diffraction C Electron density calculated from diffraction pattern D Building of a model - interpretation Scientific Background 1: Introduction

  5. Protein concentration (mg/ml) Na formate concentration (mol/L) Scientific background 2:understanding what determines crystal quality Internal structure (mosaicity, block structure) Global diffraction quality: resolution of the data Growth conditions: phase diagram

  6. The crystallization is brought about by the diffusion of a precipitant into a volume with protein solution The onset of crystallization is delayed by a spacer gel, through with the precipitant has to diffuse before reaching the protein. The diffusing precipitant can be described as a wave going through the reactor, creating different crystallization conditions in different points of the reactor. proteinsolution Precipitant volume Gel volume Scientific background 3: Counterdiffusion crystallisation Diffusion  convection-free environment is important

  7. Scientific background 4: Digital holography diagnostics(technique description see later) Measurement of the position and size of crystals Measurement of the composition of the solutions around the crystals

  8. Proposed experiment 1 Fly in parallel I one container with 23 GCB, and separately II one interferometer with six representative GCB’s Same precipitant and capillary conditions - representative GCB serve as controls for several boxes with for example different proteins, different protein concentrations, additives.  match boxes in I and II I GCF 5 weeks II ProMISS 7 days 23 reactors 6 reactors

  9. Aims: • Experimental data will be used to: • Measure residual convection and movement of the crystals – is the process purely diffusion controlled? • Verify models of the crystallisation process: Distribution of crystals in function of postition – growth conditions Depletion zone around growing crystals

  10. Proposed experiment 2:Design of the reactors • Frank Dubois, Ingrid Zegers, Emiel Van Ransbeeck, Pedeo • Exact positioning of the capillaries • Modullary design: reactor and insert are separate • Insert can be either a comb or a plate, for use with capillaries or a plate design.

  11. Proposed experiment 3:The model proteins Cab*Lys VUB Criteria for the selection of proteins: - Availability, Stability - Well established crystallisation conditions and physico-chemical properties - X-ray experiments can be done - Biological interest - Representative folds / barrels Martial Paravalbumins Declercq Collaboration by different groups on single experiments 1 Declercq Parvalbumins 2 Martial Triose phosphate isomerase 3 Wyns Cablys3 * lysozyme 4 Wyns + Garcia-Ruiz + Carotenuto Cablys3 * Lysozyme 5 Zagari (PPG)10 6 Declercq + Carotenuto Lysozyme

  12. Progress 29 september Progress 20 July Soyuz ISS 5S up 22 oct down 1 Nov. july aug sept oct nov Proposed experiment 4:Experiment Scenario Nano slab Combustion experiment Glovebox occupancy DCCO ProMISS Day 0 Day 7 A gel layer as retardant of onset of crystallisation - late access is required <X > = (D  t)1/2 • Experiment length is determined by experiment time (1 week - 3 cm max) • Delay of experiment start determined by diffusion through gel •  experiment has to be set up within days of microgravity conditions

  13. Experiment hardware (1)Basic principle Intensity image Phase image

  14. Experiment hardware (2)Basic principle Holography: Recording of the intensity and of the optical ray directions • Digital holography : • Acquisition by a CCD camera • Digital reconstruction

  15. Experiment hardware (3)Refocusing Refocusing of crystals in capillary

  16. Experiment hardware (4)Composition measurement

  17. Refocusing - movie

  18. Composition evolution - Movie

  19. Experiment hardware (5)Overview CCD camera Mechanical Accommodation to the MSG Baseplate- Thumbscrew Design.

  20. Experiment hardware (6)Overview Opening part Box: 30 cm x 30 cm 30 cm Electrical connectors Fixation by M6 Captive screws

  21. Experiment hardware (7)Top view without cover Camera + Holder Optical Subsystem 1 5 Sub-systems : Optical Subsystem 2 Experimental Wheel Laser Ground Glass

  22. Experiment hardware (8)Experimental wheel Experimental cell PromISS payload Uploaded with Progress Experimental samples uploaded With Soyuz Sample holder wheel Experimental Cells in the Sample Holder Wheel Integration by the scientist (last access before the flight)

  23. Experiment hardware (9)Travel box of the sample holder wheel with the samples Preliminary design (Hinge + latch locking) 2 Levels of containment O ring seal Foam to absorb liquid in Case of leakage of a cell Test 2 bars – 1.2Kg

  24. Experiment hardware (10)Installation of the sample holder wheel in PromISS (1) Cogged wheel DC motor Optical encoder Endless screw

  25. Experiment hardware (11)Installation of the sample holder wheel in PromISS (2) Sample holder wheel Sliding motion Rotating tray Ball lock for attachment

  26. Accommodation of PromISS and DCCO PromISS DCCO

  27. Experiment Scenario • PromISS installation • Opening of the travel box • Installation of the sample holder wheel in PromISS • Close PromISS • Activation of PromISS • Installation of a video tape (digital recorder) • Activation of PromISS • Promiss is designed to operate automatically Warm up • Video tape 1/24 hours • Download / upload of data

  28. Strong cooperation of Belgian teams on protein crystal growth, optics and industrial partners Ambitious scientific objectives Understand the fundamental processes that underly the crystallization of proteins Verify modelsfor the crystallization several model proteins PromISS is a new type of optical microscope diagnostic Refocusing without mechanical motion Accurate measurement of the composition change The crystallization of new proteins is crucial in the field of structural genomics Summary (1)

  29. PromISS involves Optical hardware (Progress) Sample holder wheel (Soyuz) PromISS operates automatically Download / upload of data Recording of the video sequences on video tapes (1/day) Summary (2)

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