Combining X-ray and Neutron Scattering Manfred Roessle EMBL Hamburg Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Birds view of the Grenoble ESRF and ILL research area. High flux reactor Institute Laue- Langevin ILL Third generation synchrotron facility ESRF Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Basic parameters Neutron reactors: • ILL Grenoble France Fission Fission Fission • FRM II Munich Germany n n n n n n n n • CARR Beijing China n n n n n n n • HFIR Oak Ridge USA Spallation Spallation Spallation Spallation sources: n n n n n n n n • PSI Villingen Switzerland n n n n n n n n n n n n • SNS Oak Ridge USA p p p n n n n • ISIS Oxfordshire UK n n n n n n n n n n n n n n n n • ESS Lund Sweden (2019) Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Neutron reactor Oak Ridge National Laboratory, Oak Ridge, USA Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Basic parameters Spectra of a cold neutron source 10 13 9 -1 ] 8 -1 sterad 7 6 -1 5 s -2 4 neutron brighness [cm The neutrons produced by reactors 3 are at too high energy (too high 2 speed, too short wavelenght …) for 10 12 9 8 scattering experiments. 7 6 5 4 They are moderated in a cold 3 2 source to lower energies. 10 11 9 8 7 6 5 4 3 9 1 9 10 7 8 2 3 4 5 6 7 8 wavelenght [ ] Wavelength [Å] Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS A classical SANS beamline D11 at the Institute Laue-Langevin ILL Grenoble France Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Basic parameters cold thermal hot Energy meV 1 25 1000 Temperature K 12 290 12000 Wavelenght nm 0.9 0.18 0.029 Velocity ms -1 440 2200 14000 Neutron mass:1.674x10 −27 kg Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Radiation from Synchrotron Storage Rings Dipole bending magnet (APS) Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Schematic SAS setup X33 BioSAXS EMBL Hamburg Beamshutter with diode for Beamstop with diode for measurement measurement of of transmitted beam incident beam (prior to exposure) 1000 mm Sample cell 1400 mm s: 0.1 nm -1 to 4.3nm -1 d : 65 nm to 15 nm Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle X-ray Scattering SAXS Basic parameters Infrared Ultraviolet X-rays Energy eV 0.1 4 12 000 Temperature K 1 1000 1 000 000 Wavelenght nm 10000 300 0.1 Velocity ms -1 300 000 300 000 300 000 Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Radiation Damage Radiation damage is caused by: • Beam heating • Hydroxyl radicals • Direct bond cracking Dissoziation of an multi- subunit protein upon X-Ray radiation damage after 10sec. Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Radiation Damage Hydroxyl radicals Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Radiation Damage Hydroxyl radicals X-ray 2 H 2 O OH ● + H 3 O - Hydroxyl radical are attacking hydrogen at the surface of proteins or DNA/RNA. DNA/RNA is very sensitive to this attack and one single hydroxyl radical cleaves the DNA/RNA. Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Contrast variation technique atomic Element or b coh for neutrons f x for X-rays number Isotope 1 H -0.374 0.28 1 D 0.667 0.28 6 C 0.665 1.69 13 C 6 0.600 1.69 7 N 0.940 1.97 8 O 0.580 2.2 5 17 O 8 0.578 2.25 12 Mg 0.530 3.38 15 P 0.510 4.23 16 S 0.285 4.50 19 K 0.370 5.30 Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Contrast variation technique From the table one can derive the following: • Neutrons are more sensitive to light atoms such as hydrogen or deuterium as X-rays • There is a large difference in the biological relevant atom hydrogen and its isotope deuterium • The b-factor does not increase with the atomic number as for X-rays Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Contrast variation technique D-labelled protein 6 [10 10 1/cm 2 ] DNA/RNA 4 H-protein 2 Buffer 0 0 20 40 60 80 fraction D 2 O in buffer [%] Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Contrast variation technique Buffer = Protein protonated (=native) protein in 40% + Buffer Protein deuterated protein in 40% Upon complex formation the proteins undergo conformational changes Mixed re-constituted complexes of d- labeled and native protein Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
The Chaperonin folding machinery main chaperonin GroEL GroES • two heptameric rings ADP • 800 kDa MW • hollow cylinder • binds denatured protein + and facilitate the refolding GroEL co chaperonin GroES • heptameric dome • 70 kDa MW • bind to one end of the GroEL cylinder and close the cavity ATP like a lid Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
GroEL/GroES complex bead modeling The in-situ structures B) B) B) A) log I(s) log I(s) log log log I(s ) I(s ) I(s ) 10 log log I(s ) I(s ) 1 0.01 0.01 0.00 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.10 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.20 0.20 0.20 0.20 0.25 0.25 0.25 0.25 0.00 0.00 0.00 0.05 0.05 0.05 0.10 0.10 0.10 0.15 0.15 0.15 0.20 0.20 0.20 0.25 0.25 0.25 s [Å -1 ] s [ Å ] s [ Å ] s [ Å ] s [Å -1 ] Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
GroEL/GroES complex Rigid body model based on the in situ A) A) B) B) Ab inito bead model structures log I(s) log I(s) log I(s ) log log I(s I(s ) ) 10 10 0.01 0.01 0.00 0.00 0.05 0.05 0.10 0.10 0.15 0.15 0.20 0.20 0.25 0.25 0.00 0.00 0.00 0.00 0.00 0.05 0.05 0.05 0.05 0.05 0.10 0.10 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.15 0.20 0.20 0.20 0.20 0.20 0.25 0.25 0.25 0.25 0.25 s [Å -1 ] s [Å -1 ] Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Intermolecular distances Stuhrmann plot Center of mass to center of mass distance detrmind by the Stuhrmann plot R. Stegmann, E. Manakova, M. Roessle & H. Heumann Journal of structural biology 1998 Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Small Angle Neutron Scattering SANS Contrast variation technique + Center to Center distance The distance of the two peaks reflects the inner molecular distance between the two visible -4 x10 Center to Center proteins in e.g. 40% distance D2O buffer solution r [ Ǻ ] 0 50 100 150 200 250 Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Combining SAXS and SANS Theoretical background One scattering data set: „Black and White“ ab inito model building, with only one the contrast solvent – particle. Multiple scattering functions with data sets from SANS contrast variation: „ Colored “ ab inito model building with multiple contrasts This approach works as well with rigid body modelling. Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Combining SAXS and SANS Protein-Protein complexes Complex formed by the receptor tyrosine kinesine Met and the Listeria monocytogenes Invasion Protein InlB. The Met extracellular region SAXS data consists of six domains: The N-terminal Sema domainis followed by a small cysteine-rich PSI domain and four immunoglobulin (Ig)-like Ig domains. InlB: 630 AS with a leucine rich repeat region with binds SANS data with high affinity to Met. Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Combining SAXS and SANS Complex of Met Receptor tyrosine kinase and Invasion Protein InlB Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
Combining SAXS and SANS Complex of Met Receptor tyrosine kinase and Invasion Protein InlB Met – InlB protein complex: Rigid body refinement of the existing high resolution structures. The Ig-like domains were kept flexible to allow refinement of the overall structure in respect to he SAXS and SANS data. H.H. Niemann, M. V. Petoukhov, M. Härtlein, M. Moulin , E. Gherardi, P. Timmins, D. W. Heinz and D. I. Svergun; J. Mol. Biol. (2008) 377, 489 – 500 Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
DNA-Protein Complex Modeling H omology modeling Ab initio modeling consider a homogenous scattering density. For proteins this is nearly always fulfilled, but not for protein-DNA/RNA SASREF modeling with homology models. complexes! Lucky case palindormic DNA! Beijing 28 th April to 6 th May 2011 EMBO Global Exchange Lecture
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