S-SAD and Fe-SAD Phasing using X8 PROTEUM

S-SAD and Fe-SAD Phasing using X8 PROTEUM Kristina Djinovic Carugo Dept. for Structural and Computational Biology Max F. Perutz Labs Univ. Vienna, Austria

Outline Fe-SAD on chlorite dismutase from Candidatus Nitrospira defluvii (NdCld) S-SAD on N-ter domain of yeast Mg channel (Mrs2)

Chlorite dismutases (Cld) Bacterial heme enzymes transform chlorite to chloride and molecular oxygen: ClO 2 Cl + O 2 Besides photosystem II, the only enzyme able to catalyze the formation of a covalent O-O bond

Clds Pentamers, about 240 residues/subunit, 1 heme per subunit NdCld expressed in E. coli, readily purified to homogeneity Cca 80% conditions in crystallization screens gave crystal hits

But Many crystal forms large number of molecules in AU (15-30) 3-6 pentamers Poor diffraction not more than 4 Å

Untill Expression of protein in hemeenriched broth Purification in presence of 1:2 molar surplus of hemin to produce fully heme b loaded protein sample, checked spectroscopically

And Diffraction to 2 Å or better, 5 molecules/au functional 5-mer Enzyme active in X-tal bubbling, i.e. producing O 2 upon addition of substrate

Solution of Phase Problem - SR Fe-SAD dataset at collected at ESRF to 2.4 Å at the Fe absorption edge Found Fe sites, but non-tracable electron density maps Gerard B.: radiation damage in Fe with effects on its Δf

Solution of Phase Problem at home X8 Proteum Wavelength (Å) 1.54 Resolution (Å) 40.7-2.40 (2.45-2.40) Space group P3 2 21 Unit cell (Å, º) a = b = 145.29, c = 135.83 Molecules / a.u. 5 Unique reflections 126515 (7108) Completeness (%) 95.5 (93.3) R meas R pim 0.0211 (0.2663) Multiplicity 24.1 (4.36) I/sig(I) 14.5 (2.2)

Substructure: cut data at 5 Å 2.1.1 SHELXC (details) NOTE : (previously) suggested high-resolution cut-off = Ã resolution 2.389 Angstroms 199.0 Friedel pairs used on average for local scaling Resl. Inf - 8.0-6.0-5.0-4.0-3.6-3.4-3.2-3.0-2.8-2.6-2.4 N(data) 1868 2482 3088 6925 5219 3598 4560 5823 7587 10134 14325 <I/sig> 46.6 42.6 41.2 38.5 30.9 27.0 21.5 16.7 10.9 7.0 3.6 %Complete 98.3 99.8 99.9 99.9 99.9 100.0 100.0 100.0 100.0 100.0 98.2 <d"/sig> 1.25 1.46 1.32 1.04 1.01 1.03 0.94 0.87 0.74 0.69 0.63 For zero signal <d'/sig> and <d"/sig> should be about 0.80 NOTE : suggesting high-resolution cut-off of 5.0 Ã NOTE : using resolution limits of 38.95-5.0 Ã

Anomalous difference Fourier Set Peak Height X Y Z # [rms] (fractional) 1 1 100.00 0.9974 0.5955 0.0192 1 2 96.10 1.1244 0.9553-0.1077 1 3 92.70 1.2405 0.8596-0.2230 1 4 92.30 1.1574 0.6335-0.1421 1 5 89.30 0.9745 0.7925 0.0446 1 6 15.20 1.0000 1.0070-0.1667 1 7 26.30 1.0918 0.6077 0.1070 1 8 24.40 1.3277 0.9550 0.0334 1 9 23.80 1.2874 0.8540-0.2247 1 10 22.90 1.2241 0.9706-0.0292 1 11 22.50 1.2871 0.6732-0.0781 1 12 21.60 1.2801 0.7476-0.0026 1 13 21.40 1.3160 0.8956-0.1104 1 14 21.30 1.1941 0.8100-0.2430 1 15 20.50 0.8912 0.7731 0.0283

Phasing and density modification with data to 2.4 Å No of Fe sites Phasing power 29 0.474 (0.150) Overall figure of merit Before dens. mod. 0.221 After dens. mod. 0.822 870 residues out of 5x241= 1205 traced, 706 docked into sequence

Maps Imidazole Experimental map at 2.4 Å Refined map at 1.85 Å

Maps Experimental map at 2.4 Å Refined map at 1.85 Å

Final NdCld:IMD Beamline ID14-2 (ESRF) Wavelength (Å) 0.933 Resolution (Å) 126.0-1.85 (1.9-1.85) Space group P3 2 21 Unit cell (Å, º) a = b = 145.67, c = 136.44 Molecules / a.u. 5 Unique reflections 268021 (18555) Completeness (%) 97.0 (90.7) R meas 0.041 (0.351) Kostan et al, J. Struct Biol. 2010 Jun 22. [Epub ahead of print] R pim Multiplicity 5.09 (5.08) I/sig(I) 24.4 (5.13) R cryst / R free 0.177 / 0.211 R.m.s.d. bonds (Å) 0.013 R.m.s.d. angles (º) 1.411

Mg transporter Mrs2 Mrs2p proteins form the major mitochondrial Mg 2+ uptake system in yeast, plants and mammalia Integral membrane protein, located in the inner mitochondrial membrane (N-terminal soluble and a C-terminal trans membrane region) 55 KDa protein, functional pentamer

Bacterial homologue structurally known periplasm Mg 2+ Mg 2+ Mg 2+ Mg 2+ Mg 2+ cytoplasm

Focus on soluble N-terminal domain Design of constructs based on bioinformatics and Ltd proteolysis 1-276 C (towards the membrane) 6 4 6 2 1 1 3 4 5 5 7 2 3 N 1-238

Ltd proteolysis Decreasing Trypsin Concentration Degraded Fractions at 4 o C Mass Spectrometry

Mapping constructs on predicted SS of Mrs2

Construct Library Constructs Residues Mrs2p 1-238 Mrs2p 1-276 Mrs2p 1-282 Mrs2p_TM1 1-305 Mrs2p_TM1, 2 1-438 Mrs2p 16-276 Mrs2p 16-278 Mrs2p 16-280 Mrs2p 16-282 Mrs2p 16-284 Mrs2p 16-286 Mrs2p 16-288 Mrs2p_BDTM1 16-305 Mrs2p BDTM1, 2 16-438

Crystals of Mrs2p(1-276) 1.7 M NaCl, 70 mm imidazole ph 7.8, at 22 C

Optimization of crystal quality of Mrs2p(1-276) 11Å 10Å 9Å 8Å 5.5Å 5.2Å 4.5Å Space Group =P6222 or P6422 Nmol/asymetric unit =6 Unit Cell= 230.0 230.0 114.47 90 90 120

Lysine methylation for crystallization of Mrs2p(1-276) 40 30 2.5 M NaCl,100 mm imidazole ph 8.0, 200 mm Zn(OAc) 2 at 22 C

Crystallization and optimization of crystal quality of Mrs2p(16-276), Mrs2p(16-278), Mrs2p(16-280) Mrs2p(16-276) Mrs2p(16-280) Mrs2p(16-278) 40% v/v Ethylene Glycol 100 mm Na/K phosphate ph 6.2 Space Group P2 1 2 1 2 1 Nmol/asymetric unit 1 Unit Cell 54.88 64.72 86.71 90 90 90 Resolution 2.6 Å Mosaicity 0.3º Completeness 99% Rmerge 7.4% Redundancy 2.6

Data-collection statistics for phasing of Mrs2p(16-276) Wave length 1.54Å Space group P2 1 2 1 2 1 Unit cell parameter a=54.66 Å, b=67.70 Å, c=85.30 Å α= = γ=90 Unique reflections 48844 Resolution range (Å) 36.89-1.83 (1.90-1.83) Completeness (%) Redundancy Anomalous completeness (%) < I >/< sigi > 98.89 (92.2) 80 (13) 92.5 (83.0) 40.23 (1.93) Measured reflections 3977702 Rmerge (%) 8.5 (88.4) Rpim (%) 0.80 (23.0)

S Substructure 10 S atoms in 260 aa residues, 1 molecule in AU 35.1-2.4 Å resolution data were used for finding S substructure

S-Substructure

Phasing, density modification, tracing to 1.83 Å Phasing Power 0.311(0.069) Figure of Merit before solvent flattening 0.201 and after solvent flattening 0.841 243 residues out of 260 were docked into the sequence

Maps Experimental map at 1.85 Å Refined map at 1.85 Å

Maps Experimental map at 1.85 Å Refined map at 1.85 Å

Maps Experimental map at 1.85 Å Refined map at 1.85 Å

Final Resolution (Å) 1.85 R (%) 20.4 α7 α5 R free (%) Number of subunits per ASU 27.5 1 α6 α3 Protein atoms 1975 α1 Water molecules 432 α4 α2 Bond lengths (Å) 0.017 Bond angles (º) 1.479 Khan, MB et al., Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010;66:658-61.

Acknowledgements Julius Kostan Beamline scientist @ ESRF Georg Mlynek Bjoern Sjoeblom Kira Gysel Claudia Schreiner Michael Wagner (UniVie) Holger Daims (UniVie) Christian Obbinger (UniVie) Paul Georg Furtmüller (UniVie) Muhammed Bashir Khan (UniVie) Rudolf Schweyen (UniVie) Christoph Romanin (BOKU)

Maps Imidazole Experimental map at 2.4 Å Refined map at 1.85 Å