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1 ISS JEM (JAXA) 2. JAXA /02/ /08/ /01/ /08/2004 Baikonur Kazakhstan Progress 03/05/ /10/ /04/ /10/2004 Kazakhstan Soyuz 13 weeks 9 weeks 13 weeks 9 weeks GCF2 GCF3 GCF3 GCF2 JCF1 GCB GCB JCB 9 GA 46 Russian Service Module GA 49 GT 20 GA 22 GT 35 GT CGBA CGBA TBU,Kryogen-3M GA Gel Acupuncture GT Gel-Tube 1
2 GT 3 GA ESA 7 CGBA TBU Kryogen-3M ESA Granada Crystallization Box GCB JAXA/JSUP Gel-Tube GCB Gel-Tube Gel-Tube 5~6 GCB 2 11 Japanese Crystallization Box JCB Gel-Tube GCB 1 JCB 2 6 GCB JCB Granada Crystallization Facility (GCF) JAXA JCF GCF1 23 GCB JCF1 12 GCB 3 4 GCF JCF 5 GCB Counter Diffusion GCB/JCB GCB/JCB ISS ISS JAXA JAXA 2
3 JAXA JAXA JAXA JAXA 4. JAXA 5. JAXA RSC RSC RSC (Raw Diffraction Data) JAXA Full sphere resolution range, R merge value, R symm value, completeness(%), I/sigma(I) 3
4 Highest sphere resolution range, R merge value, R symm value, completeness(%), I/sigma(I) JAXA JAXA JAXA ESA GCB/GCF ESA 6. ESA/ (ESA) GCF(Granada Crystallization Facility) (Federal Space Agency) This study is contributed by a part of High-quality Crystallization Project on The Protein Structure and Function Analysis for Application promoted by JAXA (Japan Aerospace Exploration Agency). GCF(Granada Crystallization Facility) that had been developed by ESA(European Space Agency) and Univ. of Granada was used for the protein crystallization, and Russian Service Module developed by Russian Federal Space Agency was used for space experiment 4
5 ) ) 5
6 X X Gel-Tube JAXA GCB 1
7 X SPring-8 Gel-Tube ( ) 2.1. JAXA ) JSUP ( ) GCF ESA RSM JAXA RSC IBMP TsPK ) ( ) GCF ) JSUP ) JSF
8 1. 2. E.coli JAXA JAXA MSDS JSUP / 1. Excel Excel 3
9 Excel JSUP 3. JSUP 4. 3 JSUP 5. JSUP JSUP 6. JSUP TEL: FAX: yamanaka-a@jsup.or.jp yamanaka@jsup.or.jp 7. JSUP PXW01674@nifty.ne.jp
10 2 (1) (2) % PEG mM 2mM 20% PEG mM 2mM PEG
11 GCB GT (Gel-Tube) JCB GT 0.5mm 0.5mm 60mm 40mm 12 l 8 l % 1mm 1% EVA 1.2mm GCB JCB 10ml 150 l/ % 90% JAXA-GCF#2~4 JAXA-GCF# (1) 1. JCB 1) 1 8 l 2) 2~6 3) 2 2. GCB 1) 1 12 l 2) 2~6 3) 2 JCB l GCB l (2) 2 JCB 2 GCB 6 40 l 170 l 6
12 Gel-Tube Gel-Tube JCB 5ml 5ml GCB 10ml 10ml JCB 10ml 10ml GCB 20ml 20ml (1) GCB 40ml 40ml 7
13 3.3. Gel-Tube JAXA JAXA JAXA 3.5. JAXA
14 Full sphere resolution range, R merge value, R symm value, completeness(%), I/sigma(I) Highest sphere resolution range, R merge value, R symm value, completeness(%), I/sigma(I) SDS-Page Native-Page DLS SDS-Page Native-Page
15 DTT PEG PEG PEG JAXA PEG 4.7. ph
16 JSUP
17 JAXA PEG
18 Crystal Data and X-ray Data Processing Statistics Diffraction data Crystals 6TAA (Swift et al.*) Ground experiment (Batch / Seeding) Space experiment (GCB)** Space experiment (GCB) X-ray source Conventional BL12B2 BL12B2 Wavelength(Å) Space Group P P P Cell constant (Å) a=51.0, b=67.2, c=133.6 = = =90º a=50.8, b=67.7, c=130.1 = = =90º a=50.4, b=67.4, c=130.4 = = =90º Volume of the cell Maximum resolution(å) Mosaicity Avarage of I/ (I) Rmerge(overall) Rmerge(outer shell) Completeness(overall)(%) Completeness (outer shell)(%) 457, , (~1.4Å) ( Å) 0.188( Å) 94.5( Å) 89.9( Å) ( Å) 0.039( Å) 99.3( Å) 99.3( Å) 442, (~0.9Å) ( Å) 0.262( Å) 96.5( Å) 95.7 ( Å) *Swift, H.J. et al.:acta Cryst., B47, (1991) **Calculated for the comparison with the data of the ground experiment. Electron Density Map of Alpha-Amylase (tentative) Crystal grown on the ground Crystal grown in space 13
19 2. PEG8000 NaCl Expected Microgravity Effects Mechanism Protein depletion zone (PDZ) formation Effects on the crystal Suppression of the cluster formation Impurity depletion zone (IDZ) formation Suppression of the step bunching Suppression of the microcrystal capture Suppression of the disorder Estimate PDZ and IDZ effects numerically in the case of alpha-amylase Diffusive field around a growing crystal In case of 30mg/ml alpha-amylase in 20% PEG8000 Ce=10mg/ml D=3.0x10 R( t) β C' e + C'( ) C'( R( t)) = D R( t) β 1+ D Concentration of the protein around the crystal surface becomes lower if the viscosity of the solution is high. s -12 m 2-1 =0.09mmhr -1 14
20 Depletion zone around the growing crystal (alpha-amylase/peg8000) Protein depletion zone Impurity depletion zone DF0 DFR = DF Ce=10mg/ml D=3.0x10-12 m 2 s -1 ß=0.09mmhr -1 G 1G C( R) Ce 1 = = C( ) Ce R β 1+ D A=10 A=20 A=50 A=100 The effects on DFR and IR are expected in case of alpa- Amylase using PEG8000 as a precipitant. IUR0 IR = IUR G 1G R β R β = D = D R βi R β A Di D βi D A = β Di Depletion zone around the growing crystal (Lysozyme/NaCl) Protein depletion zone NaCl NaCl + PEG % Ce=25mg/ml D=1.04x10-10 m 2 s -1 (NaCl) D=5.74x10-12 m 2 s -1 (NaCl+25%PEG8000) ß=0.04mmhr -1 If the viscosity of the solution is higher, the effects on DFR and IR can be expected. Impurity depletion zone NaCl NaCl + PEG % A=10 A=20 A=50 A=100 15
21 3. PEG8000 Garcia-Ruiz PDB Crystal Data and X-ray Data Processing Statistics (Tentative) 1iee *1 (Space crystal) (APCF) Ground crystal (GCB) Space crystal (GCB) X-ray source X11/BW7B BL12B2 BL12B2 Space Group P P P Cell Constants (A) a c Max Resolution(A) Mosicity ~ ~0.303 Rmerge % (total) Rmerge % (outer) 5.2(~0.94A) 6.9(~0.88A) 3.3(~1.08A) 4.0(~1.04A) 32.9( A) 25.8( A) 16.8( A) 6.6( ) Completeness % (total) Completeness % (outer) 88.6( A) 81.4( A) 83.6( A) 86.9( A) *1) Sauter et al., Acta Cryst., (2001) D57,
22 4. PEG Comparison between Maximum Resolution of Crystals grown on the ground and in space Data are collected from crystals grown in JAXA(NASDA)-GCF#1, #2, and #3 space experiments 17
23 18
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