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SIRAS Phasing:A
sample
scriptis provided below for a typical SIRAS phasing calculation.
This particular example is taken from the crystal structure of proteinase
K. All steps on the path are provided from preparing the Scalepack
files to displaying the map with O. The general path is:
scalepack2mtz ->
Example log files are given for each step, along with comments about how to judge the quality of the output. Documentation for CCP4 programs:
References for CCP4 programs:
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| #!/bin/csh
-f
# scalepack2mtz hklin ~/data/prok1.sca\ hklout mtz/nat_i.mtz << eof SYMM 96 ANOMALOUS NO END eof # # scalepack2mtz hklin ~/data/prok_io5.sca\ hklout mtz/io_i.mtz << e of SYMM 96 ANOMALOUS YES END eof # |
CCP4 document for
scalepack2mtz
The native scalepack data set, native.sca, is converted to mtz format first, followed by iodide derivative. SYMM -This keyword is compulsory and can be given as the space group name or number. Here we are using space group number 96, otherwise known as P43212. ANOMALOUS -Specify whether or not input file contains anomalous data. It is set to NO for the native, and YES for the derivatives. ---------Logfile from scalepack2mtz----------
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| #!/bin/csh
-f
truncate HKLIN mtz/nat_i.mtz \ HKLOUT mtz/nat_f.mtz \ << EOF_truncate > log/truncate_nat.log TITLE NATIVE TRUNCATE OUTPUT LABOUT F=Fnat sigF=sigFnat NRESIDUES 272 EOF_truncate # truncate HKLIN mtz/io_i.mtz \ HKLOUT mtz/io_f.mtz \ << EOF_truncate > log/truncate_io.log TITLE NATIVE TRUNCATE OUTPUT LABOUT F=Fio sigF=sigFio DANO=Dio SIGDANO=sigDio NRESIDUES 272 EOF_truncate |
CCP4 document for
truncate
The native and derivative data sets are processed with truncate. Truncate converts the intensities into structure factors by taking the square root. Hence, the input files have the extension _i.mtz and the output files have the extension _f.mtz. It also puts the data sets on an absolute scale. This makes it convenient for estimating heavy atom occupancies. LABOUT -Output labels for each column of data. These should be meaningful and easy to remember. NRESIDUES -The number of residues in the asymmetric unit. Used to put the data sets on an absolute scale. ---------Logfile from truncate---------- Truncate outputs some useful statistics. The Wilson B-factor is calculated and plotted: SCALE = exp( - intercept). Least squares straight line gives: B = 21.434 SCALE = 216.53 where F(absolute)**2 = SCALE*F(observed)**2*EXP(-B*2*SINTH**2/L**2) |
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| #!/bin/csh
-f
cad hklin1 mtz/nat_f.mtz \ hklin2 mtz/io_f.mtz \ hklout mtz/derivs_unscaled.mtz \ <<eof-cad RESOLUTION OVERALL 90.0 1.8 SYMMETRY 96 TITLE native and Iodide derivative SCAL FILE 1 1.0 LABIN FILE 1 E1=Fnat E2=sigFnat CTYP FILE 1 E1=F E2=Q LABOUT FILE 1 E1=Fnat E2=sigFnat LABIN FILE 2 E1=Fio E2=sigFio E3=Dio E4=sigDio
END
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CCP4 document for
cad
The native and iodide derivative data sets are collected into one single file with cad. LABIN- Input labels for each column of data. LABOUT -Output labels for each column of data. These should be the same
as LABIN.
---------Logfile from cad---------- |
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| #!/bin/csh
-f
# scaleit hklin mtz/derivs_unscaled.mtz \ hklout mtz/derivs.mtz << eof >log/scaleit.log TITLE Scaling native and derivatives GRAPH H K L MODF REFINE ANISOTROPIC LABI FP=Fnat SIGFP=sigFnat - FPH1=Fio SIGFPH1=sigFio - DPH1=Dio SIGDPH1=sigDio eof cat log/scaleit.log
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CCP4 document for
scaleit
The derivative is scaled to the native data set using an overall scale factor and B factor. Grep for the word "acceptable" from the log file to see what threshold should be used for the exclude statement in mlphare.com. ---------Logfile from scaleit---------- |
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| #!/bin/csh
-f
# mlphare HKLIN mtz/derivs.mtz\
ATOM1 I- -0.0559 0.2496 0.2872 1.0 1.0 BFAC
25.0
ATOM2 I- 0.2518 0.1552 0.3514 1.0 1.0
BFAC 25.0
ATOM3 I- 0.5913 0.2789 0.1858 1.0 1.0
BFAC 25.0
ATOM4 I- -0.0435 0.2228 0.4155 1.0 1.0 BFAC
25.0
ATOM5 I- -0.0148 0.4773 0.0318 1.0 1.0 BFAC
25.0
ATOM6 I- -0.0910 0.0769 0.2634 1.0 1.0 BFAC
25.0
ATOM7 I- 0.0191 0.2712 0.3975 1.0 1.0
BFAC 25.0
ATOM8 I- 0.4869 -0.1185 0.4652 1.0 1.0 BFAC
25.0
ATOM9 I- 0.5524 -0.0890 0.1452 1.0 1.0 BFAC
25.0
ATOM10 I- 0.172 -0.0129 0.0698 1.0 1.0 BFAC
25.0
ATOM11 I- -0.138 0.1329 0.3132 1.0 1.0 BFAC
25.0
ATOM12 I- 0.1027 -0.1027 0.250 1.0 1.0 BFAC
25.0
ATOM13 I- 0.0206 0.0992 0.0891 1.0 1.0 BFAC
25.0
END-phare
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CCP4 document for
mlphare
Plug in the iodide positions from ShelxD. Refine heavy atom positions for 15 cycles and calculate phases using both isomorphous and anomalous differences. Because ocupancy and Bfactor parameters are coupled, refinement cycles should alternate between occupancy and bfactor refinement as implemented in this script. Refinement of both occupancy and Bfactor during the same cycle can be unstable. I usually include the highest resolution possible in the phasing calculation. Although the statitistics will be poor for the highest shells, Dm will improve the phases with solvent flattening. ---------Logfile from mlphare---------- Mlphare outputs some useful statistics. Check that the refinement parameter and anomalous refinement parameter are converging toward 1.0 with each cycle of refinement. If not, then further cycles of refinement may be necessary (not usually the case.) ---------------------------------------------
Check the phasing power (PhP_a and PhP_c), these should be 1.0 or greater
for resolution shells below 3.0 Angstrom resolution. Check that RCullis
is under 1.0
This includes the isomorphous difference between
FP and FPH,
Unweighted MEAN lack-of-closure analysis
TOTAL 19226 51.7 45.5 1.18 0.88 3230 72.8 64.9 1.04 0.89 *******************************************************
This includes the anomalous difference, the expected
Unweighted MEAN anomalous lack-of-closure analysis
TABLE: Anomalous lack of closure analysis v resln using MEANS- Phasing cycle - deriv 1: Number_acentric_reflections
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| dm
hklin mtz/derivs_ph.mtz\
hklout mtz/dm.mtz \ << my-data >log/dm.log SOLC 0.40 MODE SOLV HIST MULT RESOLUTION 20 1.8 COMBINE omit NCYCLE AUTO #SCHEME RES FROM 2.0 LABIN FP=Fnat SIGFP=sigFnat - PHIO=PHIO FOMO=FOMO - HLA=HLA HLB=HLB HLC=HLC HLD=HLD LABOUT PHIDM=PHI1 FOMDM=W1 - HLADM=HLADM HLBDM=HLBDM - HLCDM=HLCDM HLDDM=HLDDM my-data |
CCP4 document for
Dm
If your native data set extends to higher resolution than the derivative,
then use phase extension to the highest resolution possible. High
resolution phases are important for success in ARP/wARP automated chain
tracing.
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| #!/bin/csh
-f
#calculate fourier map for viewing # fft HKLIN mtz/dm.mtz \ MAPOUT map/prok.map << END_fft RESO 20 1.8 LIST 100 FFTSPG 1 TITL prok Fourier map phase with I-. BINMAPOUT XYZLIM 0 1 0 1 0 .5 LABI F1=Fnat SIG1=sigFnat PHI=PHI1 W=W1 SCALE F1 1.0 0.0 END_fft # # extend MAPIN map/prok.map \ MAPOUT map/prok.ext << END-extend XYZLIM 0 1 0 1.0 0 1 SYMMETRY 96 END-extend rm fftkw.abcoeffs al_mapman -b mapsize 10000000<<eof
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CCP4 document for
fft
CCP4 document for extend convert map to dsn6 format for display in O. Also calculate a bones representation of the density for viewing with O. Here is a convenient macro for viewing your map with O.
map_file prok.omap
read_fo prok.odb
menu @mapmove on
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