S-SAD and Fe-SAD Phasing using X8 PROTEUM

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1 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

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

3 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

4 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

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

6 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

7 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

8 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

9 Solution of Phase Problem at home X8 Proteum Wavelength (Å) 1.54 Resolution (Å) ( ) Space group P Unit cell (Å, º) a = b = , c = Molecules / a.u. 5 Unique reflections (7108) Completeness (%) 95.5 (93.3) R meas R pim (0.2663) Multiplicity 24.1 (4.36) I/sig(I) 14.5 (2.2)

10 Substructure: cut data at 5 Å SHELXC (details) NOTE : (previously) suggested high-resolution cut-off = Ã resolution Angstroms Friedel pairs used on average for local scaling Resl. Inf N(data) <I/sig> %Complete <d"/sig> 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 Ã

11 Anomalous difference Fourier Set Peak Height X Y Z # [rms] (fractional)

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

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

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

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

16 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

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

18 Focus on soluble N-terminal domain Design of constructs based on bioinformatics and Ltd proteolysis C (towards the membrane) N 1-238

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

20 Mapping constructs on predicted SS of Mrs2

21 Construct Library Constructs Residues Mrs2p Mrs2p Mrs2p Mrs2p_TM Mrs2p_TM1, Mrs2p Mrs2p Mrs2p Mrs2p Mrs2p Mrs2p Mrs2p Mrs2p_BDTM Mrs2p BDTM1,

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

23 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=

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

25 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 P Nmol/asymetric unit 1 Unit Cell Resolution 2.6 Å Mosaicity 0.3º Completeness 99% Rmerge 7.4% Redundancy 2.6

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

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

28 S-Substructure

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

30 Maps Experimental map at 1.85 Å Refined map at 1.85 Å

31 Maps Experimental map at 1.85 Å Refined map at 1.85 Å

32 Maps Experimental map at 1.85 Å Refined map at 1.85 Å

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

34 Acknowledgements Julius Kostan Beamline 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)

35

36

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

38

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