::!\bin\sh :: ============================================================ :: Outline of Heavy atom refinement and Phasing tutorial :: ( E.J.Dodson) :: ============================================================ :: Example based on MIR solution of Dendrotoxin from green mamba :: Protein of 425 residues :: cell 73.58 38.73 23.19 90.00 90.00 90.00 :: Spacegroup P212121 :: Two derivatives; Au I :: Steps after solving patterson for 1 site. :: Use FFT, PEAKMAX HAVECS, Int Tab etc.. ::======================== :: step 1. ::======================== :: Refine Au Site 1 using Centric differences only :: Phasing cycle ( the last) will calculate SIR phases for all reflections. :: Alternative: Use a direct methods program to find a consistent set of Hg :: sites and go straight to Step 3. :: :: Step 2. ::======================== :: Difference Fourier to find more sites. Contour and :: note pertubation in map about first site. :: Check patterson peaks are consistent with 2nd site. ( do not worry :: too much if they are not... (gets round alternate origin problem) :: :: Step 3. ::======================== :: Refine 2 Au Sites using Centric differences only :: Phasing cycle ( the last) will calculate SIR phases for all reflections :: :: Step 4. ::======================== :: Difference fouriers to look for I sites. :: :: Step 5. ::======================== :: Refinement of both derivatives together. :: :: Step 6. ::======================== :: Refinement of both derivatives together including anomalous data :: If hand correct, and there are TWO independent derivatives; :: anomalous occupancies will be positive if hand correct; :: negative if hand incorrect. :: If so - change positions to -x,-y,-z, ( spacegroup to :: enantiomorph if necessary) and go on.. :: ::======================== :: Next steps not in this example: :: :: Step 7. ::======================== :: Check I pattersons. :: Now should repeat procedure, using I phases to find Au. :: Calculate isomorphous map... :: :: Step 8. ::======================== :: Calculate isomorphous map... :: :: Step 9. ::======================== :: Do density modification. (dm\Wang procedures) :: Calculate new map... :: :: Step 10. ::======================== :: Build structure. :: :: :::::: Command procedures to carry out this procedure. :: ================================================== :: STEP 1 :: ================================================== :: step 1. :: Refine Au Site 1 using Centric differences only :: Phasing cycle ( the last) will calculate SIR phases for all reflections. ::---------------------------------------------------- ::---------------------------------------------------- :: step1_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- IF NOT EXIST %TEMPRES%\toxd_sc.mtz (echo '! run scaleit-ex first' 1>&2 && GOTO :EOF) mlphare HKLIN %TEMPRES%\toxd_sc.mtz HKLOUT %TEMPRES%\toxd_step1.mtz < %SCRIPTWIN%\mir-steps1.dat :: ================================================== :: STEP 2 :: ================================================== :: :: Step 2. :: Difference Fourier to find more sites. Contour and :: note pertubation in map about first site. :: Check patterson peaks are consistent with 2nd.. sites. (do not worry :: too much if they are not...) :: (If you add FH=FH1 PHIH=PHIH1 you will generate a double difference map :: - may be clearer..) ::---------------------------------------------------- ::---------------------------------------------------- :: step2_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- :: fft HKLIN %TEMPRES%\toxd_step1.mtz MAPOUT %TEMPRES%\toxd_step2.map abcoeffs %TEMPRES%\fftkw.abcoeffs < %SCRIPTWIN%\mir-steps2.dat :: Find 100 peaks above 5*rms, :: output to PDB file "occupancy"= peak height, " Btemp"= height\sigma :: orthogonalization code (normal) :: positive peaks only peakmax mapin %TEMPRES%\toxd_step2.map xyzout %TEMPRES%\toxd_step2.frc < %SCRIPTWIN%\mir-steps3.dat :: ================================================== :: STEP 2A :: ================================================== :: :: Get PDB file of coordinates and vectors. :: Alternative inputs - PDB,fractional or phare_ml :: 1) Input fractional sites :: Free format: Occup x y z b (sites.frc - typed in) :: :: 2) Input PHARE fractional sites ::ATOM Au 0.026 0.000 0.287 19.75 25.74 BFAC 13.887 :: :: 3) Input PDB file - usual sort of stuff CRYSTAL\SCALEi coordinates. :: havecs XYZIN %DATA%\sites.frc XYZOUT %TEMPRES%\sites.pdb UVWOUT %TEMPRES%\sitesuvw.pdb < %SCRIPTWIN%\mir-steps4.dat :: Plot map with Au sites marked. Choose sensible contour levels :: ::!\bin\csh -f npo MAPIN %TEMPRES%\toxd_step2.map XYZIN1 %TEMPRES%\sites.pdb PLOT %TEMPRES%\step2.plot < %SCRIPTWIN%\mir-steps5.dat :: ================================================== :: STEP 3 :: ================================================== :: :: Step 3. ::======================== :: Refine 2 Au Sites using Centric differences only :: Phasing cycle ( the last) will calculate SIR phases for all reflections :: ::---------------------------------------------------- ::---------------------------------------------------- :: step3_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- :: mlphare HKLIN %TEMPRES%\toxd_sc.mtz HKLOUT %TEMPRES%\toxd_step3.mtz DATOUT test.dat < %SCRIPTWIN%\mir-steps6.dat ::================================================== :: STEP 4 ::================================================== :: :: Step 4. ::======================== :: Difference fouriers to look for I sites and Hg sites ::---------------------------------------------------- :: step4a_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- :: In this case it was important to EXCLUDE ridiculous differences. :: ::!\bin\csh -f fft HKLIN %TEMPRES%\toxd_step3.mtz MAPOUT %TEMPRES%\step4.map abcoeffs %TEMPRES%\fftkw.abcoeffs < %SCRIPTWIN%\mir-steps7.dat peakmax MAPIN %TEMPRES%\step4.map XYZOUT %TEMPRES%\siteI.frc < %SCRIPTWIN%\mir-steps8.dat ::---------------------------------------------------- ::---------------------------------------------------- :: step4b_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- :: :: ::!\bin\csh -f fft HKLIN %TEMPRES%\toxd_step3.mtz MAPOUT %TEMPRES%\step4b.map abcoeffs %TEMPRES%\fftkw.abcoeffs < %SCRIPTWIN%\mir-steps9.dat peakmax MAPIN %TEMPRES%\step4b.map XYZOUT %TEMPRES%\siteHg.frc < %SCRIPTWIN%\mir-steps10.dat ::================================================== :: STEP 5 ::================================================== :: :: Step 5. ::======================== :: Refinement of all derivatives together. :: Include refinement of anomalous occupancies. ::---------------------------------------------------- ::---------------------------------------------------- :: step5_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- mlphare HKLIN %TEMPRES%\toxd_sc.mtz HKLOUT %TEMPRES%\toxd_step5.mtz DATOUT %TEMPRES%\test.dat < %SCRIPTWIN%\mir-steps11.dat :: ================================================== :: STEP 6 :: ================================================== :: Refinement of both derivatives together. :: Include refinement of anomalous occupancies on both hands. ::---------------------------------------------------- ::---------------------------------------------------- :: step6_mir_steps ::---------------------------------------------------- ::---------------------------------------------------- mlphare HKLIN %TEMPRES%\toxd_sc.mtz HKLOUT %TEMPRES%\toxd_step6.mtz DATOUT %TEMPRES%\test.dat < %SCRIPTWIN%\mir-steps12.dat ::---------------------------------------------------- ::---------------------------------------------------- :: step6a_mlphare.com ::---------------------------------------------------- ::---------------------------------------------------- :: only necessary for certain spacegroups -- dummy step here mtzutils HKLIN1 %TEMPRES%\toxd_sc.mtz HKLOUT %TEMPRES%\scaled.mtz < %SCRIPTWIN%\mir-steps13.dat mlphare HKLIN %TEMPRES%\scaled.mtz HKLOUT %TEMPRES%\toxd_step6.mtz DATOUT %TEMPRES%\test.dat < %SCRIPTWIN%\mir-steps14.dat