s-sad and fe-sad phasing using x8...

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:

ClO2¯ → Cl¯ + O2

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 heme-enriched 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 O2 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) 1.54Resolution (Å) 40.7 - 2.40

(2.45 - 2.40)Space group P3221Unit cell (Å, º) a = b = 145.29,

c = 135.83Molecules / a.u. 5Unique reflections 126515 (7108)Completeness (%) 95.5 (93.3)Rmeas

Rpim 0.0211 (0.2663)Multiplicity 24.1 (4.36)I/sig(I) 14.5 (2.2)

Substructure: cut data at 5 Å2.1.1 SHELXC (details)

NOTE : (previously) suggested high-resolution cut-off = Ã…

resolution 2.389 Angstroms199.0 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 - 2.4N(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 FourierSet Peak Height X Y Z# [rms] (fractional)1 1 100.00 0.9974 0.5955 0.01921 2 96.10 1.1244 0.9553 -0.10771 3 92.70 1.2405 0.8596 -0.22301 4 92.30 1.1574 0.6335 -0.14211 5 89.30 0.9745 0.7925 0.04461 6 15.20 1.0000 1.0070 -0.16671 7 26.30 1.0918 0.6077 0.10701 8 24.40 1.3277 0.9550 0.03341 9 23.80 1.2874 0.8540 -0.22471 10 22.90 1.2241 0.9706 -0.02921 11 22.50 1.2871 0.6732 -0.07811 12 21.60 1.2801 0.7476 -0.00261 13 21.40 1.3160 0.8956 -0.11041 14 21.30 1.1941 0.8100 -0.24301 15 20.50 0.8912 0.7731 0.0283

Phasing and density modification with data to 2.4 Å

870 residues out of 5x241= 1205 traced, 706 docked into sequence

No of Fe sitesPhasing power

290.474 (0.150)

Overall figure of meritBefore dens. mod. 0.221After dens. mod. 0.822

Maps

Experimental map at 2.4 Å Refined map at 1.85 Å

Imidazole

Maps


FinalNdCld:IMD

Beamline ID14-2 (ESRF)Wavelength (Å) 0.933Resolution (Å) 126.0-1.85

(1.9 - 1.85)Space group P3221Unit cell (Å, º) a = b = 145.67,

c = 136.44Molecules / a.u. 5Unique reflections 268021 (18555)Completeness (%) 97.0 (90.7)Rmeas 0.041 (0.351)Rpim

Multiplicity 5.09 (5.08)I/sig(I) 24.4 (5.13)Rcryst/ Rfree 0.177 / 0.211R.m.s.d. bonds (Å) 0.013R.m.s.d. angles (º) 1.411

Kostan et al, J. Struct Biol. 2010 Jun 22. [Epub ahead of print]

Mg transporter Mrs2

Mrs2p proteins form the major mitochondrial Mg2+ 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

cytoplasm

Mg2+Mg2+

Mg2+

Mg2+Mg2+

Focus on soluble N-terminal domain

Design of constructs based on bioinformatics and Ltd proteolysis

6

N

C (towards the membrane)

1

2

13

2 3

4

5

6

7

4

5

1-238

1-276

Ltd proteolysis

Mass Spectrometry

Decreasing Trypsin Concentration Degraded Fractions at 4oC

Mapping constructs on predicted SS of Mrs2

Construct LibraryConstructs 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

1.7 M NaCl, 70 mM imidazole pH 7.8, at 22°C

Crystals of Mrs2p(1-276)

11Å10Å

9Å8Å5.5Å5.2Å4.5Å

Space Group =P6222 or P6422Nmol/asymetric unit =6 Unit Cell= 230.0 230.0 114.47 90 90 120

Optimization of crystal quality of Mrs2p(1-276)

2.5 M NaCl,100 mM imidazole pH 8.0, 200 mM Zn(OAc)2 at 22°C

30

40

Lysine methylation for crystallization of Mrs2p(1-276)

40% v/v Ethylene Glycol100 mM Na/K phosphate pH 6.2

Mrs2p(16-276) Mrs2p(16-280)

Mrs2p(16-278)

Space Group P212121Nmol/asymetric unit 1 Unit Cell 54.88 64.72 86.71 90 90 90Resolution 2.6 ÅMosaicity 0.3ºCompleteness 99%Rmerge 7.4%Redundancy 2.6

Crystallization and optimization of crystal quality of Mrs2p(16-276), Mrs2p(16-278), Mrs2p(16-280)

Wave length 1.54Å

Space group P212121

Unit cell parameter a=54.66 Å, b=67.70 Å, c=85.30 Åα=β= γ=90

Unique reflections 48844

Resolution range (Å) 36.89 - 1.83 (1.90-1.83)

Completeness (%) 98.89 (92.2)

Redundancy 80 (13)

Anomalous completeness (%)

92.5 (83.0)

< I >/< sigI > 40.23 (1.93)

Measured reflections 3977702

Rmerge (%) 8.5 (88.4)

Rpim (%) 0.80 (23.0)

Data-collection statistics for phasing of Mrs2p(16-276)

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


Final

α1

α2

α3

α4

α6

α5

α7

Resolution (Å) 1.85

R (%) 20.4

Rfree (%) 27.5

Number of subunits per ASU 1

Protein atoms 1975

Water molecules 432

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

s-sad and fe-sad phasing using x8...

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