Structural investigations of redox regulation in ATFKBP13 3

Chapter Materials and Methods CHAPTER 3. MATERIALS AND METHODS 3.1 EXPRESSIONS AND PURIFICATION OF ATFKBP13 The mature AtFKBP13 gene, which codes 129 amino acids, was cloned into the prokaryotic expression vector pGEX-KG [Rajeev Gupta et al., 2002]. The protein was over expressed in E. coli BL21 (DE3) cells. The cells were grown in Luria-Bertani medium to an OD600 of 0.6 at 310 K and expression of the recombinant protein was induced with 0.5 mM isopropyl -D-thiogalactopyranoside (IPTG) at 303 K. Cell growth was continued at 303 K for h after IPTG induction and cells were harvested by centrifugation at 4,200g (6000 rpm, Beckman JA-8.1000 rotor) for 10 at 277 K. The cell pellet was suspended in ice-cold lysis buffer [20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1mM DTT] and homogenized by sonication. The crude lysate was centrifuged at 42, 400g (18,000 rpm, Beckman JA-25.50 rotor) for h at 277 K and the cell debris was discarded. The supernatant was applied to a GST-affinity column (5 ml glutathione Sepharose 4B) and the contaminant proteins were washed away with wash buffer (lysis buffer plus 400 mM NaCl) and eluted with 50 mM Tris (pH 7.5), 10 mM reduced glutathione. 150 units of thrombin were added to the eluate and incubated overnight at 277 K. The fusion protein was cleaved efficiently (Fig. 3-1). The eluate was dialyzed in 20 mM Tris (pH 7.5), 0.5 M NaCl using 3,500 Da molecular weight cut-off dialysis tubing (Spectra). Thrombin and the cleaved GST were removed by passing through a 55 Chapter Materials and Methods ml HiTrap benzamidine FF (Pharmacia) column and a ml GSTrap FF column. The final purification step was achieved by gel filtration on a HiLoad 16/60 Superdex-75 prep-grade column (Pharmacia) previously equilibrated with a buffer solution containing 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The purified protein was concentrated to 10 mg ml-1 using an YM10 membrane (Amicon) and confirmed with Bradford protein assay [Bradford, 1976]. The protein was then aliquoted as 50 µl per tube, flash frozen with liquid nitrogen and stored at –80 ºC for later use. The purified protein was analyzed on SDS-PAGE and native PAGE. The dynamic light-scattering data showed the protein had 70-80% homogeneity as a monomer. M 58.1 40.0 29.0 20.1 14.3 Figure 3-1. SDS-Page showing the purification of recombinant AtFKBP13 using Glutathione matrix. M- Marker, Lane 1- Lysate, Lane 2- 56 Chapter Materials and Methods Flow-through, Lane 3- Wash, Lane 4- Eluate, Lane 5- Thrombin cleaved, Lane 6- FPLC purified. 3.2 CHLOROPLAST IMPORT ASSAYS AND PROTEIN LOCALIZATION A radiolabeled AtFKBP13 precursor protein was synthesized by a coupled transcription and translation procedure in wheat germ extract, in the presence of [35S]methionine and [35S]cysteine. Chloroplasts were isolated from pea and incubated with the precursor protein as described [Mould and Gray,1998]. Import assays containing intact chloroplasts (0.5 mg chlorophyll ml-1), mM methionine, mM cysteine, and 10 mM MgATP in a final volume of 500 µl of import buffer [50 mM Hepes•KOH (pH 8.0), 0.33 M sorbitol] and 45 µl of products from in vitro translation were incubated in light (100 µmol photons m–2 s–1) for 45 min. For protein import in the presence of nigericin or sodium azide, isolated chloroplasts were incubated with µM nigericin or 10 mM sodium azide for 10 on ice. The 35 S-labeled precursor protein was then added and samples were incubated at 25 °C for 25 in light. After import, protein samples were analyzed by electrophoresis on 20% polyacrylamide gels in the presence of SDS followed by fluorography. For protein localization, Arabidopsis chloroplasts were isolated from protoplasts according to an earlier method [Sommerville et al., 1981]. The stromal and luminal fractions were analyzed by western blotting to determine the distribution of AtFKBP13 (and plastocyanin) in these two fractions. 57 Chapter 3.3 Materials and Methods IN VITRO PROTEIN–PROTEIN INTERACTION ASSAYS The purified precursor or mature form of AtFKBP13 protein was mixed with GST-Rieske fusion proteins (1:1 molar ratio), immobilized on glutathione beads in a final volume of 500 µl containing binding buffer [50 mM Tris•HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 20, mM PMSF]. After gentle shaking for 1.5 h at 22 ºC, the beads were pelleted and washed three times with the binding buffer. Proteins were eluted by 10 mM glutathione, resolved by SDS/PAGE, and detected by western blot analysis. 3.4 PROTEIN EXTRACTION AND WESTERN BLOT ANALYSIS Total proteins from leaves of 4-week old plants were extracted in a buffer [50 mM Tris (pH 7.5), 100 mM NaCl, mM EDTA, 0.1% Triton X-100, mM DTT, mM PMSF, mM benzamidine, µg ml-1 leupeptin, µg ml-1 aprotinin, µg ml-1 pepstatin A]. The homogenate was mixed for 15 s, incubated on ice for min, and centrifuged at 12,000g for 10 at °C. The supernatant was collected, and proteins were quantified by using the Bradford assay kit (Bio-Rad). For western blot analysis, 30 µg of total proteins were separated in 12% SDS polyacrylamide gel and proteins were transferred onto nitrocellulose membrane. Bound antibodies were detected with a chemiluminescence kit (Amersham Pharmacia). 3.5 FKBP13 REDUCTION BY THIOREDOXIN Reduction experiments were performed using recombinant AtFKBP13 purified after cleavage by thrombin. Reduction by the NADP/thioredoxin system of Escherichia 58 Chapter Materials and Methods coli was performed as described [Wong et al., 2003]. Recombinant AtFKBP13 (1.5 µg) was incubated with 0.25 mM NADPH, 0.3 µg NADP-dependent thioredoxin reductase (NTR), and 0.3 µg thioredoxin in 50 mM Tris-HCl (pH 7.5) at 25 °C for 20 min. Newly exposed cysteines resulting from disulfide reduction were labeled with the addition of thiol-specific fluorescent probe monobromobimane (mBBr) to mM. After labeling, the protein sample was separated by SDS-PAGE [Laemmli, 1970] and the fluorescence recorded using Gel Doc-1000 fitted with a UV 365 nm transilluminator and the Quantity One analysis program (Biorad). Subsequently, the protein pattern was revealed by staining with Coomassie Blue G-250 and captured using a scanner. 3.6 PPIASE ASSAY All assays were carried out using the GST-AtFKBP13 fusion protein. GST alone showed no PPIase activity, and the fusion protein showed no reduction of PPIase activity compared to the thrombin-cleaved pure protein. PPIase assay was performed using the protocol of Kofron et al. (1991), with modifications. 45 nM enzyme was incubated with 1.5 mg α-chymotrypsin in reaction buffer [50 mM HEPES (pH 8.0), 100 mM NaCl] and the reaction was allowed to stabilize to 10 °C. AtFKBP13 was reduced by incubation with 0.5 µM of chloroplast m-type or E. coli thioredoxin and 500 µM DTT for 20 at 25 °C. The synthetic peptide Suc-Ala-Ala-Pro-Phe-paranitroanilide was dissolved in 470 mM LiCl in trifluoroethanol to maximize the amount of peptide present as the cis-isomer. The reaction was started by adding peptide substrate to a final concentration of 60 µM and the catalysis was monitored at 390 nm in a Cary 3E UV/visible spectrometer (Varian) 59 Chapter Materials and Methods and data were obtained with the Kinetics application. kcat/Km values were calculated as kobs-k0/[PPIase], where k0 represents the first order rate constant for spontaneous cis-trans isomerisation [Liu et al., 1990]. 3.7 BIOPHYSICAL PROPERTIES OF ATFKBP13 3.7.1 Circular dichroism spectroscopy CD spectroscopy is a monitor of the overall protein secondary structure and is sensitive to conformational changes [Drake, 1994]. For CD studies, recombinant AtFKBP13 was prepared, which was eluted as a single peak from the HiLoad 16/60 Superdex-75 gel filtration column. The protein were then dialyzed against phosphate buffer of varying pH. The secondary structures of the above protein under different pH conditions were examined using their CD spectra. Each CD spectrum showed a large negative differential molar extinction coefficient between 210 nm and 220 nm, with a small trough between these wavelengths, as expected for proteins with α-helix and βsheet contents [Drake, 1994]. These characteristics of the native CD spectra are lost at pH 2.5, where the protein is in the substantially unfolded state. Fig. 3-2 shows the Far-UV spectra of the native state and the unfolded state of this protein. The AtFKBP13 at pH 7.0 and at pH 6.0 spectra are very similar, and indicate that they have similarly folded structures. They are readily distinguishable from AtFKBP13 at pH 2.5, which shows more of random coil. The protein seems to be more stable at pH 4.0 but reveals a reduction of β-strand by 4%. 60 Chapter Materials and Methods Figure 3-2. CD spectra AtFKBP13 at 20 ºC and varying pH conditions. The differential molar extinction coefficient is shown as a function of wavelength. The sample concentration was 1.5 mg ml-1. Color codes used: blue (pH 8.5), red (pH 7.0), yellow (pH 6.0), brown (pH 4.0), and green (pH 2.5). These results were obtained in three CD sessions with three independent preparations. These results suggest that the protein at physiological acidic pH is active and retains its secondary structure. 61 Chapter 3.7.2 Materials and Methods Mass spectrometry The precise mass of the protein was determined by MALDI TOF-MS (Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry) here using a Voyager-DE™ Biospectrometry™ workstation equipped with a 337-nm nitrogen laser. To obtain a good signal-to-noise ratio, 150-200 single shot spectra were collected. Saturated sinapinic acid in 50 % acetonitrile was used as the matrix. The fractionated protein (0.5 µL) was mixed with 0.5 µL of the matrix and dried on 96 × sample holder prior to the analysis. The molecular weight was determined to be 13,527 ± 1.07 Da (Fig. 3.3). This showed that the protein was >95% pure. Figure 3-3. Mass Spectrometry for AtFKBP13 62 Chapter 3.8 Materials and Methods CRYSTALLIZATION AND DATA COLLECTION 3.8.1 Crystallization of AtFKBP13-S2 Initial screening of crystallization conditions followed the sparse-matrix sampling method [Jancarik and Kim, 1991] using Crystal Screen (Hampton Research) and Protein Crystallography Basic Kit (Sigma-Aldrich). Crystallization was performed using the hanging-drop vapor-diffusion method [McPherson, 1990] at 293 K using a 24-well VDX plate (Hampton Research). The size of the droplet, which consisted equal volumes of AtFKBP13-S2 (oxidized AtFKBP13) and reservoir solution, was µl. After d, clusters of twinned plate-like `sheaves' or needle-like crystals were found in three conditions at 2.0 M ammonium sulfate, 5% v/v isopropanol (Crystal Screen II, No. 5), 0.1 M HEPES (pH 7.5), 2% PEG 400, 2.0 M ammonium sulfate (Crystal Screen I, No. 39) and 0.1 M trisodium citrate (pH 5.6), 20% v/v isopropanol, 20% w/v PEG 4000 (Crystal Screen I, No. 40). Figure 3-4. AtFKBP13-S2 crystal 63 Chapter Materials and Methods These conditions were used as the starting points for optimization experiments using selected reagents from Additive Screens (Hampton Research) and varying the buffer, temperature and protein concentration. Single crystals (Fig. 3-4) were grown at 293 K in 100 mM Tris (pH 7.9), 8-11% PEG 550 MME, 2.5 M ammonium sulfate. Nucleation occurred within days and crystals reached their maximum size in approximately two weeks. 3.8.2 Crystallization of AtFKBP13-(SH)2 Crystals of reduced AtFKBP13 [AtFKBP13-(SH)2] were produced in the same way as the oxidized AtFKBP13 crystals. The protein was maintained in the reduced form throughout, over a period of two months, by the addition of DTT after crystallization. 3.8.3 Data collection and analysis Prior to data collection, single crystals were rapidly swept through mother liquor containing 20% (v/v) glycerol as a cryoprotectant and were flash-frozen in liquid nitrogen at 100 K. Frozen crystals were screened at 100 K using an in-house X-ray facility (Rigaku RU-H3R rotating-anode X-ray generator operated at 50 kV and 100 mA with an R-AXIS IV imaging-plate detector). Diffraction data for AtFKBP13-S2 and reduced AtFKBP13 were collected using synchrotron radiation at 100 K (Oxford Cryostream). All diffraction intensities were integrated and scaled using the HKL software package [Otwinowski and Minor, 1997]. The crystal data information is given Table 3-1. 64 Chapter Materials and Methods Table 3-1. Crystal parameters, data-collection and processing statistics. Values in parentheses are for the highest resolution shell, (1.92-1.85) and (1.95-1.88 Å) respectively. AtFKBP13-S2 AtFKBP13-(SH)2 Space group C2221 C2221 a (Å) 89.026 88.898 b (Å) 126.606 125.753 c (Å) 119.404 119.424 Matthews coefficient (Å3/Da) 2.5 2.5 Percentage solvent 50.3 50.3 No. of molecules in ASU NSLS, BNL (X12B) APS (17-ID) Detector ADSC Q315 CCD ADSC Q210 CCD Resolution (Å) 1.85 1.88 Total observations 199040 3,190,736 Unique reflections 53,794 54,269 Completeness (%) 93.6 (71.3) 99.8 (98.4) Redundancy 3.7 7.2 0.080 (0.537) 0.089 (0.621) Unit-cell parameters Data collection X-ray source Rsym Rsym = ∑hkl∑i[|Ii(hkl) – | / Ii(hkl)] 65 Chapter 3.9 Materials and Methods STRUCTURE DETERMINATION 3.9.1 Molecular replacement candidates Sequence alignment with AtFKBP13 as well as spectroscopic studies suggests that FKBPs are structurally homologous. After eliminating the NMR structures and the structure of complexes, three possible candidates, human native Fkbp [PDB code: 1D6O, Burkhard et al., 2000]; yeast Fkbp [PDB code: 1YAT Rotonda et al., 1993]; Bos taurus Fkbp12 [PDB code: 1FKK, Wilson et al.,1995] were considered as probes for molecular replacement. The statistics of each candidate are presented in Table 3-2. Table 3-2. Statistics of Fkbp probe candidates Candidate PDB Resolution, R- Sequence Sequence code Å value identities positives 1D6O 2.2 16.2% 32.71% 46.5% 1YAT 2.5 17.7% 38.93% 48.00% 1FKK 2.20 15.8% 32.71% 42.6% Human native Fkbp Yeast Fkbp Bos taurus Fkbp12 From the alignment we see that all candidates share high sequence identities with AtFKBP13. The structural homology was also confirmed by making a three-dimensional 66 Chapter Materials and Methods superposition of the given structures using the Dali program [Holm and Sander, 1993]. The root mean square deviation (r.m.s.d.) of the Cα-atom positions in all three structures is within 0.7 Å, confirming that each of these structures can be used as a molecular replacement probe. However, yeast FKBP was chosen as the search probe since it has a slightly higher percentage of positive sequence match. 3.9.2 Molecular replacement of AtFKBP13 The calculated solvent content of the oxidized AtFKBP13 crystal indicates that AtFKBP13 molecules are present in the asymmetric unit. Different combinations of resolution limits, integration radii, and temperature factor distributions were tested for molecular replacement. Table 3-3. Translation function solutions for AtFKBP13-S2. α, β, γ are Eulerian angles within the AMoRe conventions. tx, ty, tz are fractional translations; R = R-factor. Solution α β γ tx ty tz cc R 1-5 97.73 54.86 135.65 0.497 0.136 0.393 0.160 0.584 2-6 130.0 58.90 140.81 0.749 0.179 0.870 0.570 3-1 169.9 55.71 132.96 0.682 0.677 0.394 0.2550 0.552 4-1 19.66 56.55 140.87 0.034 0.416 0.377 0.295 0.543 5-5 31.13 29.99 77.24 0.541 0.207 0.659 0.600 0.970 0.303 67 Chapter Materials and Methods A rotational search using the 20 to Å data in AMoRe [Navaza, 1994 ] resulted in a set of 10 peaks with correlation coefficients larger than half of that of the first peak. The translation search revealed the true nature of these peaks. The translation function was performed on the best rotational peaks using the 20 - 3.0 Å resolution range data. The height of the produced peaks was limited to half the height of the maximum peak. The final output had a correlation coefficient of 30.3% and an R-factor of 54.1% (Table 3-3). Rigid body fitting of these peaks improved the solution considerably. This rigidbody refinement is considered to be another checking procedure, to prove the correctness of the solutions. The least-squares minimization, with respect to the rotational and positional parameters, is performed for each molecule while the others are kept fixed. The rigid-body refinement of all the AtFKBP13 molecules yielded the final correlation coefficient of 35.7% and R-factor of 49.2% for 20 to Å data. These values confirmed that these solutions were correct. 3.9.3 Molecular replacement of AtFKBP13-(SH)2 AtFKBP13-(SH)2 was solved by molecular replacement using the MOLREP program (Vagin and Taplyakov, 1997) and the oxidized AtFKBP13 structure as the probe. The Key active residues (Cys5, Cys17, Cys106 and Cys111) were mutated to alanine to prevent any model bias. The final model had an R-factor of 35.7% and a correlation value of 63.6%. 68 Chapter 3.10 Materials and Methods STRUCTURE REFINEMENT The ARP/wARP [Lamzin et al., 2001] software suite was used for automation of model building and refinement using the molecular replacement solution. The output of warpNtrace contained 80 to 98 % polypeptides fragments. Main-chain tracing and building was performed using XtalView [McRee, 1999]. The remainder of the structure (cis-prolines, poorly ordered loops and terminal residues for each fragment) was manually completed using the O program [ Jones et al., 1991]. 3.10.1 Structure refinement of AtFKBP13 Refinement of AtFKBP13 started with the calculation of 2Fobs-Fcalc and Fobs-Fcalc maps using the molecular replacement solutions. The first refinement started from an Rfactor of 0.49 with the atomic temperature factors fixed set at 20 Å2. In the initial stages, only the atomic coordinates were refined. The restraints on geometry were adjusted by adapting the relative weight of the contribution of the X-ray data. At the last stages, temperature factors were allowed to refine isotropically and water was picked-up in five cycles of the water-pick up program of CNS. The model was checked for stereochemical correctness using the programs PROCHECK and WHAT_CHECK [Laskowski et al., 1991; Hooft et al., 1996]. All stereochemical parameters were flagged as either within normal limits or better when compared to a structure at this resolution. There are two regions in the protein with noticeably high temperature factors. These include both the N and C termini loop regions. 69 Chapter Materials and Methods We suggest that these loop regions may undergo a conformational change when the Cterminal active site cysteins are reduced. The error spikes in the PROCHECK [Laskowski et al., 1993] output became smaller in subsequent runs after the introduction of information suggested by the program during the rebuilding stages. This indicated more geometrically acceptable changes are incorporated and refinement is progressing in the right direction. Refinement of the water molecules was made easier and more successful with when the automatic refinement program ARP [Lamzin and Wilson, 1992]. 3.10.2 Refinement of the reduced structure Later it became clear that the N-terminus of the AtFKBP13-(SH)2 was poorly defined, after which many omit maps were calculated (by setting the occupancy of appropriate atoms to zero and performing a few cycles of refinement) and rebuilding was attempted. To ensure that the structure was correct, a series of simulated-annealing refinement was undertaken, followed by omit map calculations. Refinement was carried out for each of the alternate conformations individually and also by co-refining them. The final R-factor and other refinement statistics for both the molecules are given in Table 34. 70 Chapter Materials and Methods Table 3-4. Refinement parameters AtFKBP13-S2 AtFKBP13-(SH)2 Resolution range (Å) 8–1.85 8–1.88 Reflections (working/test) 40,309/4,530 40,687/4,569 0.21 / 0.23 0.20 / 0.23 Non-hydrogen atoms 4,630 4,630 Waters 463 313 Protein 21.497 32.367 Waters 32.060 39.379 R.M.S.D. in bond lengths (Å) 0.007 0.008 R.M.S.D. in bond angles (°) 1.7 1.6 Rcryst / Rfree Final model: Average B-factors (Å2): R-factor = ∑hkl||Fo(hkl)| – |Fc(hkl)|| / ∑hkl|Fo(hkl)| The oxidized and reduced structures were superimposed and r.m.s.d calculations were performed using the program O [Jones et al.,1991]. Coordinates of AtFKBP13-S2 and AtFKBP13- (SH2) have been deposited at the PDB with accession codes 1U79 and 1Y00 respectively. 71 [...]... 34 70 Chapter 3 Materials and Methods Table 3- 4 Refinement parameters AtFKBP 13- S2 AtFKBP 13- (SH)2 Resolution range (Å) 8–1.85 8–1.88 Reflections (working/test) 40 ,30 9/4, 530 40,687/4,569 1 0.21 / 0. 23 0.20 / 0. 23 Non-hydrogen atoms 4, 630 4, 630 Waters 4 63 3 13 Protein 21.497 32 .36 7 Waters 32 .060 39 .37 9 R.M.S.D in bond lengths (Å) 0.007 0.008 R.M.S.D in bond angles (°) 1.7 1.6 Rcryst / Rfree Final model:... alanine to prevent any model bias The final model had an R-factor of 35 .7% and a correlation value of 63. 6% 68 Chapter 3 3.10 Materials and Methods STRUCTURE REFINEMENT The ARP/wARP [Lamzin et al., 2001] software suite was used for automation of model building and refinement using the molecular replacement solution The output of warpNtrace contained 80 to 98 % polypeptides fragments Main-chain tracing... fixed The rigid-body refinement of all the AtFKBP 13 molecules yielded the final correlation coefficient of 35 .7% and R-factor of 49.2% for 20 to 3 Å data These values confirmed that these solutions were correct 3. 9 .3 Molecular replacement of AtFKBP 13- (SH)2 AtFKBP 13- (SH)2 was solved by molecular replacement using the MOLREP program (Vagin and Taplyakov, 1997) and the oxidized AtFKBP 13 structure as the probe... percentage of positive sequence match 3. 9.2 Molecular replacement of AtFKBP 13 The calculated solvent content of the oxidized AtFKBP 13 crystal indicates that 5 AtFKBP 13 molecules are present in the asymmetric unit Different combinations of resolution limits, integration radii, and temperature factor distributions were tested for molecular replacement Table 3- 3 Translation function solutions for AtFKBP 13- S2... Main-chain tracing and building was performed using XtalView [McRee, 1999] The remainder of the structure (cis-prolines, poorly ordered loops and terminal residues for each fragment) was manually completed using the O program [ Jones et al., 1991] 3. 10.1 Structure refinement of AtFKBP 13 Refinement of AtFKBP 13 started with the calculation of 2Fobs-Fcalc and Fobs-Fcalc maps using the molecular replacement... angles within the AMoRe conventions tx, ty, tz are fractional translations; R = R-factor Solution α β γ tx ty tz cc R 1-5 97. 73 54.86 135 .65 0.497 0. 136 0 .39 3 0.160 0.584 2-6 130 .0 58.90 140.81 0.749 0.179 0.870 0.570 3- 1 169.9 55.71 132 .96 0.682 0.677 0 .39 4 0.2550 0.552 4-1 19.66 56.55 140.87 0. 034 0.416 0 .37 7 0.295 0.5 43 5-5 31 . 13 29.99 77.24 0.541 0.207 0.659 0.600 0.970 0 .30 3 67 Chapter 3 Materials... incorporated and refinement is progressing in the right direction Refinement of the water molecules was made easier and more successful with when the automatic refinement program ARP [Lamzin and Wilson, 1992] 3. 10.2 Refinement of the reduced structure Later it became clear that the N-terminus of the AtFKBP 13- (SH)2 was poorly defined, after which many omit maps were calculated (by setting the occupancy of appropriate... performing a few cycles of refinement) and rebuilding was attempted To ensure that the structure was correct, a series of simulated-annealing refinement was undertaken, followed by omit map calculations Refinement was carried out for each of the alternate conformations individually and also by co-refining them The final R-factor and other refinement statistics for both the molecules are given in Table 34 ...Chapter 3 Materials and Methods Table 3- 1 Crystal parameters, data-collection and processing statistics Values in parentheses are for the highest resolution shell, (1.92-1.85) and (1.95-1.88 Å) respectively AtFKBP 13- S2 AtFKBP 13- (SH)2 Space group C2221 C2221 a (Å) 89.026 88.898 b (Å) 126.606 125.7 53 c (Å) 119.404 119.424 Matthews coefficient ( 3/ Da) 2.5 2.5 Percentage solvent 50 .3 50 .3 No of molecules in. .. was limited to half the height of the maximum peak The final output had a correlation coefficient of 30 .3% and an R-factor of 54.1% (Table 3- 3) Rigid body fitting of these peaks improved the solution considerably This rigidbody refinement is considered to be another checking procedure, to prove the correctness of the solutions The least-squares minimization, with respect to the rotational and positional . weeks. 3. 8.2 Crystallization of AtFKBP 13- (SH)2 Crystals of reduced AtFKBP 13 [AtFKBP 13- (SH)2] were produced in the same way as the oxidized AtFKBP 13 crystals. The protein was maintained in the. (working/test) 40 ,30 9/4, 530 40,687/4,569 1 R cryst / R free 0.21 / 0. 23 0.20 / 0. 23 Final model: Non-hydrogen atoms 4, 630 4, 630 Waters 4 63 3 13 Average B-factors (Å 2 ): Protein 21.497 32 .36 7. completed using the O program [ Jones et al., 1991]. 3. 10.1 Structure refinement of AtFKBP 13 Refinement of AtFKBP 13 started with the calculation of 2F obs -F calc and F obs -F calc maps using