Experimental Phasing stuff

Centric reflections |F P | |F PH | FHFH Isomorphous replacement F P + F H = F PH FPFP F PH FHFH

Phase ambiguity for acentric reflections Isomorphous replacement |F PH | - |F P |  |F H | FHFH F PH FPFP

Anomalous dispersion F HKL  F HKL F PH + F PH -* FHFH FP+FP+ F PH + F PH - FHFH FP+FP+ FP-FP- F Hano + F Hano -

Combine SIR & SAD Phase ambiguity in SAD is exactly perpendicular to SIR F PH + F PH -* FHFH FP+FP+ FHFH FPFP F PH SAD SIR SIRAS is more powerful than SAD / SIR alone

DENZO/SCALEPACK Processing MOSFLM/SCALA Estimation of ANO/ISO diffs SCALEPACKSCALEITSHELXCXPREP..XTRIAGE Find HA sites SHELXD RSPSSOLVE HYSS Crosscheck sites FFTRSPSMLPHARE SnB.. Refine sites SHARPSHELXD/E SOLVE Density modification SHELXE RESOLVE DMSOLOMON CNS XDS.. PIRATE..

Native data, 2.8Å, P6 3 [188.64, , 45.86] Au derivative data, 3.5Å, P6 3 [188.76, , 46.2] Xyloglucan endotransglycosylase, XET, 30kDa 1% cell differences ~ 25% intensity change at 3 Å

SCALEIT, native vs derivative DISODANO How do these differences compare with the errors of F P and F PH ?

 Real = |F PH | - |F P |  2 FPH +  2 FP  Real  Expect Possible derivative ; slope > 4 intercept > 0.1 Nearly identical ; slope  1 intercept  0 Normal probability analysis of DISO Compare isomorphous differences to a normal distribution

Normal probability analysis (SCALEIT) Gradient & intercept plotted against resolution

SHELXC, anomalous signal? A value of d”/sig > 1.3 usable

FFT, anomalous difference Patterson

FFT, difference Patterson

SHELXD, input file Sensitive parameters; Resolution Number of HAs

SHELXD, logfile

Check your sites before SHARP!

MLPHARE

Switch to MLPHAREs coordinate system..

Crosscheck sites

||F PH  F P | - F Hcalc | |F PH  F P |  R cullis cen = < 1 interesting < 0.9 useful |F Hcalc | |F PHobs  F PHcalc |  PP cen =  > 1 interesting > 1.2 useful Performance measures for experimental phasing Centric reflexes 

Phase probability curve m = |F P | best |F P | observed F best Figure of merit (FOM) Gives an estimate of the uniqueness of the solution Center of gravity gives best approximation of FP FOM = 0.5 → 60° phase error

Anomalous R cullis for first site

Three top sites..

Difference Fourier map

Sidestep ; density modification in DM

SHARP Compound Crystal Wavelength Batch

SIRAS case

MAD case

f’ and f” values critical

Add parameters slowly

End of 2 nd SHARP run

Find more sites?

Hand 1 Hand 2

Hand 1 Hand 2

SOLOMON DM

Ready for O, averaging, ArpWARP etc..