Amyloid Precursor Protein (APP) Unknown function Proteolysis generates beta amyloid peptide Alzheimer disease patients: imbalance in homeostasis copper and zink Proteolytic processing of APP is influenced by metal ions APP ectodomain contains two domains: E1 and E2 E1 domain: growth factor- like domain and copper binding domain (CuBD) The fold og E2 domain is highly flexible; disordered, anisotropic crystals with high overall B- factors NMR studies: only a part of the E2 domain is rigidly folded in solution

Methods Crystallization: sitting- drop vapor diffusion; drop 1:1 (10 mg/mL protein:buffer) Tetragonal crystals obtained Crystallization conditions: 0.1 mM HEPES (pH 6.4), 1M sodium acetate, 10 mM MgCl2 and 50 mM CdSO4, R(-)-2-methyl-2,4-pentanediol Competetive metal binding experiments: crystals soaked with 10 mM CuCl2 or ZnCl2 Phasing: MR with Caenorhabditis elegans APL-1 E2 structure as search model and cadmium SAD low- energy data set

Patterson function An autocorrelation function of the electron density! Patterson map: map of interatomic distance vectors Patterson function is the convolution of the electron density at any point u=(u, v, w) over the whole unit cell in real space R: The explicit form useful for numeric evaluation: Used for Substructure solution Molecular replacement searches The Patterson function can be computed from experimental data; F2=I and phases = 0.

Molecular replacement Intramolecular Patterson vectors: depend on orientation of the molecule Intermolecular Patterson vectors: depend both on orientation and position in the unit cell Search model: identical / homologus protein, components of complex, individual domains 1) Patterson rotation searches: 2) Patterson translation searches  The Cheshire cell : minimum cell that needs to be covered to obtain an unique solution

SAD: single- wavelength anomalous dispersion X- ray absorption of selected elements f’: dispersive component: intensity differences at different wavelengths f’’: anomalous component: intensity differences between Bijvoet pairs in one dataset Lambert- Beer absorption law:

SAD: single- wavelength anomalous dispersion Friedel’s law: Friedels law breaks down in the presence of anomalous scatterers! Peak: maximum f’’ Inflection point: minimum of f’ High- energy remote: f’ close to 0 Low- energy remote: f’ and f’’ close to 0 Anomalous Argand diagram Harker diagram FPA= FP+ FA+ iF’’A

SAD dataset Space group: P43

Composite annealed 2Fo-Fc omit map contoured at 0.9𝜎 Zink- specific anomalous double difference map: 7𝝈 Anomalous difference map at low energy: 5𝝈 Copper- specific anomalous double difference map: 7𝝈 Anomalous difference map at low energy: 5𝝈

Conclusion E2 domain highly flexible Metal ion co-crystallization: better diffraction Cadmium ion binds unspecifically in multiple states Sites with high specificity for zink and copper Conformational changes upon binding of different metal ions