2. 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

3. 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

4. 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.

5. 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

6. 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:

7. 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

8. SAD dataset
Space group: P43

9. 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

10. 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