1. First principle computations X-ray spectra & diffraction patterns
A synergic approach to the structural study of metallo-protein complexes
Francesco Stellato
I.N.F.N. Roma ‘Tor Vergata’
Milan, Jan 12th 2017

2. Summary Intro: < 5 minutes for 5 W’s
Metal ions, protein misfolding & neurodegenerative diseases
In vitro: the Experimental side
Serial crystallography at synchrotrons & FELs
X-ray Absorption at synchrotrons & FELs
In silico: the Computational side
Classical molecular dynamics & ab initio calculations
X-ray Absorption & Diffraction simulations
Budget & Timeline

3. What?

4. In words… Protein mis-folding  aggregation fibrils
*** Experiments
XRD & XAS  structure and dynamics
*** Theory
Interpret data in terms of molecular structures & aggregates
A- (proteins + metals + water )  classical MD
B- (metal binding)  QM calculations
C- Compute XRD and XAS from QM electron density calculations
Aβ, PrP Metal ions (Cu, Zn)

5. Comprehensive picture of complex phenomena
Why?
High-Performance Computers
& soft-matter
theoretical
models
Brilliant X-rays
(Synchrotrons & Free Electorn Lasers - FELs)
Connecting two worlds
Comprehensive picture of complex phenomena

6. Who? 10 years experience in
-XAS and XRD Experiments at synchrotrons and FELs in Europe and in the US
-Developing novel experimental techniques: serial crystallography
High performance computing: MD &
in silico simulation of in vitro experiments

7. When? The time is now ! High-performance computing resources
Novel FEL and synchrotron X-ray sources
Novel FEL and synchrotron X-ray techniques
2000s – Growth of amyloyd nanocrystals
2010 – First X-rays Free Electron Lasers
2011 – First XRD FELs
2012 – First X-ray FELs
2014 – First serial synchrotrons
2015 – Ab initio calculation of XAS data
2015 – Revival of quantum XRD
2017 – Availability of “cheap” HPC time

8. Where? INFN Rome “Tor Vergata” Synchrotron (ESRF, Petra III, SLS)
Two young scientists directly involved in the project:
1 PhD student working 70% on the project (E. de Santis)
1 Researcher working 30% on the project (V. Minicozzi)
Expertise to build, host and run a mid-sized HPC cluster is available on site & environment with strong expertise in X-ray measurements and computational methods (and in combining them):
2 INFN staff (R. Ammendola & G. Salina)
2 Full Professors (S. Morante & G.C. Rossi)
> 20 years activity CSN4 and/or CSN5
Synchrotron
(ESRF, Petra III, SLS)
& Free Electron Laser (FEL)
(European XFEL, LCLS)
sources around the world

9. Protein Folding
Protein folding is strictly connected to protein functioning. Mis-folding is responsible of Protein Conformational Disorders
Even unrelated mis-folded proteins (with homologies neither in sequence nor in structure) build up fibrils with remarkably similar morphology and cross-beta structure

10. Metals, Mis-Folding & Neurodegeneration Transmissible Encephalopaties
Alzheimer’s Disease
A peptide plaques in a mis-folded, fibrillar form
Abnormally high metal concentration
Transmissible Encephalopaties
The cellular prion protein folding moves from a cellular form (PrPC) into an aberrant conformer (PrPSc)
Cu and Zn are thought to have different effects on peptide oligomerization, aggregation and fibril formation
The process is influenced by the presence of metal ions (mainly Cu and Zn)
Minicozzi, Stellato et al., JBC 2008
Stellato et al., EBJ 2006
De Santis, JPCB 2015
Morante et al. JBC 2004; Stellato et al., EBJ 2011; Stellato et al., EBJ 2014

11. Experimental Techniques
X-Ray Diffraction (XRD)
Atomic resolution structures of whole macromolecule,
BUT needs crystals
Radiation-damage limited resolution
X-Ray Absorption Spectroscopy (XAS)
Atomic resolution structures ONLY in the vicinity of metal ions, BUT does not need crystals

12. Serial Femtosecond X-ray Crystallography
Serial: each crystal gives a diffraction pattern
Femtosecond: diffraction-before-destruction to overrun radiation damge
Crystallography: crystals as small as 300 nm
TheX-raydoseneededtoachieveagivenresolutionforaparticularsamplecanbecalculated.Itcanbeshownthatthedoserequiredtoimageasinglebiologicalmoleculeismuchlargerthanthedoserequiredtocompletelydestroythemoleculethrghradiationdamageprocesses.X-raycrystallographersmitigatethisproblembyspreadingthedamageoverbillionsofmoleculesinasinglecrystal,greatlyenhancingthediffractionsignal.Sincethemoleculesareallidenticalandpreciselyalignedinthecrystal,theX-rayscatteringinformationispreservedandthestructurecanbedetermined.
LCLSoffersanotherwayaroundthedamageproblem.SincetheFELX-raypulseisveryintenseandveryshort,itispossibleinprincipletodelivertherequireddosetoanano-scalesampleandrecordthescatteredX-rayinformationbeforethedamageprocesseshavetimetodestroythesample.Inotherwords,anLCLSX-raypulsecouldbefocusedontoasinglemolecule,whichwouldbedestroyed–butnotbeforethescatteredX-raysarealreadyontheirwaytothedetectorcarryingtheinformationneededtodeducetheimage.TheCoherentX-rayImaging(CXI)Instrumentwillofferthepossibilityofdeterminingstructuresatresolutionbeyondthedamagelimitforsampleswhichdonotformcrystals,includingimportantclassesofbiologicalmacromolecules
Thousands of patterns are recorded and need to be analyzed
an HPC challenge
Free Electron Laser
(FEL)
1013 photons/30 fs
FEL beam
Chapman et al., Nature 2011

13. Serial Crystallography at Synchrotrons
Using a synchrotron for serial XRD allows spreading the dose over thousands of crystals
Micro-crystals, too small for conventional technqiques, can provide useful diffraction
Synchrotron
1013 photons/1 s
Stellato et al., IUCrJ 2014

14. Brewster et al. Acta Cryst D 2015
Research Plan / 1
1- Grow amyloid
nano- & micro-crystals in different conditions (pH, metal ions)
Brewster et al. Acta Cryst D 2015
Nelson et al., Nature 2005
2- Perform (parallel and simultaneous)
XAS & XRD measurements (both at synchrotrons & FELs)

15. Research Plan / 2
serial XRD: high-resolution structures from nano- and micro-crystals
Serial XAS: high-resolution structures of the metal binding site
Comparison between metal binding sites in crystalline (XRD) and non-crystalline (XAS) form
Sample
Experimental Technique
Deliverables
A & prion microcrystals
Synchr. serial crystallography
High-res electron density
A & prion nanocrystals
FEL serial crystallography
A & prion fibrils w/o metal ions
Microscopy, AFM
Fibril conformation
Non-crystalline A & prion
XAS
Metal binding site structure
Crystalline A & prion

16. Classical and ab initio calculations
Classical Molecular Dynamics Simulations
ideal tool to simulate large molecules, but unable to give information on bond formation/breaking
Ab initio Calculations (Quantum Mechanics) computationally expensive, but allow to reliably calculate electron densities and analyze bond formation and breaking

17. Ab initio XAS calculations
Initial State
Final state with
a core-hole
Initial state:
no core-hole
Incident-photon
energy
Final State
La Penna, …, Stellato, J Phys Chem 2015

18. Ab initio XRD calculations
Standard X-ray crystallography methods use free-atom models to calculate mean unit-cell charge densities.
Real molecules have charge densities not accurately captured by the routinely used free-atom models
Charge density of crystalline urea - Wall , IUCrJ 2016
Charge density of crystalline molecules calculated using Density Functional Theory and compared with experimental data

19. Research Plan / 3
Comparison of structural data from XAS & XRD experiments and calculations
Sample
Methodology
Deliverables
Amyloid multimers (>10 monomers)
Classical MD
Peptide aggregation
Amyloid monomers and dimers
Ab initio methods
Metal binding site structure
XAS spectra and XRD patterns ab initio calculations
Computational and experimental data comparison
Amyloid with and without metal ions
X-ray damage (Coulomb explosion dynamics)
Simulation of damage process (Coulomb explosion) and comparison with XAS & XRD experimental data

20. XAS spectra and XRD pattern calculations:
Computational needs
Classical MD simulations of 10 A peptides or prions in complex with metal ions in a water environment ( 105 atoms):
0.5x106 core hours +
Ab initio calculations for fragments of A peptides or prions in complex with metal ions ( 103 atoms  3x103 electrons ):
106 core hours
XAS spectra and XRD pattern calculations: =
2x106 core hours

21. INFN Rome ’Tor Vergata’
HPC cluster
A suitable machine
128 cores  128x24x365 = 1 MCh / year
Gives flexibility in HPC time usage
and in software optimization
dual CPU Intel E5-2697AV4
(4 nodes, each 16x2 2.6 GHz) 256 GB RAM DDR TB HD storage
Infiniband FDR Frontend node, RAID controller  
Switch Infiniband: FDR (56 Gb/s)
To be hosted in the
APE-lab
INFN Rome ’Tor Vergata’

22. Timeline
The computational part will start immediately using already available (although limited) computational resources
Proposal writing for beamtime access will start immediately.
The experiments will start as soon as the samples will be available

23. Budget
Computational side
€  128 cores parallel computing = 1 MCh/year
Experimental side €
€ amyloid proteins ( 400 €/mg)
5.000 € wetlab consumables
Travel expenses
€ access to light source facilities
5.000 € participation to conferences and workshops
Other costs €
4.000 € open access publications
6.000 € running costs of the cluster € =
€ year 1 +
€ year 2

24. Conclusion Novel computational methods, high computing power
Novel X-ray sources,
novel techniques
People,
Expertise,
Ideas
Ideas need to be powered… €€€
Pure science: getting closer to understand neurodegeneration
Applied science: drug design
- Methods: techniques development
- Cross-fertilization: experimental & computational scientists interaction

25. Thank you
for your attention

26. Research Plan / 4
Classical MD & ab initio calculations of Aβ and prions with and without metal ions
Calculation of XAS spectra and XRD pattern coming from structures along the MD trajectories  taking into account the structural disorder
Atomic configuration C1
XAS1
XRD1
Atomic configuration C2
XAS2
XRD2
Average XAS spectrum
& XRD pattern … …
Atomic configuration Cn
XASn
XRDn

27. Computations & Experiments
Molecular dynamics (classical and ab initio):
strucuture of Aβ & prions in the absence and in the presence of metal ions
Several experimental techniques have been used to assess the role of metal ions in A & prions mis-folding and aggregation
This project will exploit and combine in vitro & in silico X-ray Absorption Spectroscopy and X-ray Diffraction with Molecular Dynamics

28. Diffract-and-destroy experiments
One pulse,
one measure
Particle injection
A detectable signal must be recorded before the sample is destroyed
FEL 1013 photons/30 fs vs
Synchrotron 1013 photons/s
The diffract-and-destroy idea can work for in principle all synchrotron
X-ray techniques
FEL puse
Diffraction pattern
R. Neutze et al, Nature 406 (2000)