Anything input on a line after "!" or "#" is ignored and lines can be continued by using a minus (-) sign. The program only checks the first 4 characters of each keyword. The order of the cards is not important except that an END card must be last. Some keywords have various subsidiary keywords. The available keywords in this section are:
- Input MTZ labels
- Number of the refinement cycles
- Refinement parameters
- Scale parameters
- Parameters of the likelihood (sigmaA)
- Parameters of the solvent
- Weighting X-ray vs geometry
This keyword tells the program which columns in the MTZ file should be used as native structure factors, sigmas, FreeR flag, phase information etc.
-------------------------------------------------------------------------- # # Only native structure factors, their sigmas and FreeR_flag # are given # LABIn FP=F_native SIGFP=SIGF_native FREE=FreeR_flag # or # # Apart from native structure factors, their sigmas and FreeR_flag # some phase information in a form of Hendrickson and Lattman # coefficients also known. It gives signal to the program that # phased refinement should be used # LABI FP=F_native SIGF=SIGF_native FREE=FreeR_flag - HLA=HLA_phases HLB=HLB_phases HLC=HLC_phases HLD=HLD_phases --------------------------------------------------------------------------
LABIn is essential for all refinement except geometry idealisation. To some extent the course of the refinement is governed by the assignments given. The following program labels can be assigned:
FP SIGFP FREE FPARTi PHIPi HLA HLB HLC HLD or PHIB FOM
This keyword controls the type of refinement or idealisation.
------------------------------------------------------------------------------ # ####Restrained refinement. Reflections between 20 - 1.5Å will be used # REFI TYPE RESTrained RESOLUTION 20 1.50 # # Use maximum likelihood residual # REFI RESI MLKF # # Refine individual isotropic B values REFI BREF ISOTropic or REFI TYPE REST RESO 20 1.50 REFI RESI MLKF BREF ISOT REFI METH CGMAT or # # Rigid body refinement # REFI TYPE RIGID #(all other definitions are defaults) ------------------------------------------------------------------------------
In more detail, these subkeywords are:
REFInement TYPE RESTrained
This keyword describes the type of refinement.
REFInement PHASed SCBLur 1.0 BBLUr 0.0
If experimental phases are being used it may be necessary to blur the phase probabilities, especially after some density modification calculations (this information can also be input with the keyword PHASE).
Program will apply blurring as follows:
HLAnew = HLA*scblur*exp(-(sin(theta)/lambda)**2*bblur) HLBnew = HLB*scblur*exp(-(sin(theta)/lambda)**2*bblur) HLCnew = HLC*scblur*exp(-(sin(theta)/lambda)**2*bblur) HLDnew = HLD*scblur*exp(-(sin(theta)/lambda)**2*bblur)
or if PHASE and FOM are given: the program first generates the Hendrickson-Lattman coefficients using the formula:
HLA = Func(FOM)*COS(DEGTOR*PHASE), HLB = Func(FOM)*SIN(DEGTOR*PHASE), HLC = HLD = 0.
i.e. the Phase probability distribution is unimodal.
REFInement RESIdual MLKF
This keyword describes the Xray part of the function.
Fxray = SUM(Whkl*(|FO|-|FC|)**2)
Fxray = SUM(LLKcentric_hkl) + SUM(LLKacentric_hkl)
If experimental phase information is available the residual is modified appropriately. This is invoked by assigning appropriate input columns; see LABIN (for methodology see G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997) in Acta Cryst. D53, 240-255, or Pannu, Murshudov, Dodson and Read (1998) in Acta Cryst. D54, 1285-1294).
REFInement METHod CGMATrix
This keyword describes method of minimisation.
REFInement BREFinement ISOTropic
This keyword describes method for parameterisation of atomic Bvalues (atomic displacement parameters).
Default: Use all reflections
Include all well measured data, not omitting the weak observations; it will be weighted appropriately. The low resolution data helps define the solvent shell. However if you have lost strong terms by some accident of data collection, the scaling may not behave well.
REFInement TLSCycles 0
It controls scaling of calculated and observed structure factors. The SCALE keyword has several different options. See below for keywords for estimation of sigmaA, triggered by SCALe MLSC. For example:
# Use Babinet's bulk solvent type scaling # SCALe TYPE BULK # and/or # # do anisotropic scaling. Use resolution between 100 and 2.1Å # SCALe LSSC ANIS RESO 100 2.1 # # Use simple scaling, i.e. do not use Babinet's bulk solvent # SCALe TYPE SIMP # # Fix B value of Babinet's bulk solvent. It is useful when # bulk solvent based on the constant value is used. SCALe LSSC FIXBulk BBULk 200
In more detail, these subkeywords are:
with one of the following sub-subkeywords:
KB = K0*exp(-B0*s^2) * (1- K1*exp(-B1*s^2))
The scale formulation is based on the Babinet principle and described by Dale Tronrud and others. Better results can be obtained if bulk solvent correction based on a constant value is used. See SOLVENT.
KB = K0*exp(-B0*s^2) (Simple Wilson scaling) i.e. K1 = 0
This may be more appropriate if keyword SOLVENT is active.
Lower resolution structures may not have sufficient data to give a robust Wilson plot overall B factor, so it is possible to fix the <B> for the structure to a set value. If you are using this option it is important to add remaining B-value to observed structure factors.
[Default all data are used for the scaling]
Defines resolution limit for scaling.
APPL OBSE | CALC will apply overall Bcorrection to either observed or calculated structure factors.
Flag to indicate all following subkeywords apply to estimation of scale between Fo and Fc.
Lower resolution structures may not have sufficient data to find sensible
overall scales and B values for both the BULK and the protein.
SCBULK = <solvent_density>/<protein_density>
i.e. For aqueous solvent, with solvent density ~ 1.0. and protein density ~ 1.35, SCBULK ~ 1.0/(1.35). If bulk solvent based on a constant value (SOLVENT) is used then fixing of BBULK is necessary. In this case SCALE TYPE SIMPLe also could be used.
Many crystals generate seriously "anisotropic" reflection data. This is presumably due to some crystalline disorder, and is not the same as anisotropy of individual atoms. However the correction can be expressed in a similar form.
Then, apart from isotropic overall B factor B0, contribution of anisotropic B centered at the origin of coordinate system (i.e. in orthogonal system (B11+B22+B33 = 0.0) is also refined.
Overall anisotropic B values are applied to the calculated structure factor with Miller index h,k,l as follows:
B11*h*h*(a*)^2 + B22*k*k*(b*)^2 + B33*l*l*(c*)^2 + 2.0*B12*h*k*(a*)*(b*) + 2.0*B13*h*l*(a*)*(c*) + 2.0*B23*k*l*(b*)*(c*) where h,k,l are Miller indices a*,b*,c* are reciprocal space cell dimensions
REFMAC estimates overall anisotropic B values only once at the first cycle and keeps them constant for the rest of the REFMAC refinement session. For R, free are calculation contribution of them is applied to the calculated structure factor. During refinement it is applied to the observed structure factor.
Anisotropic scaling of data should ideally be done at the merging stage but often the distortion aligns with the crystal axes, and therefore cannot be detected from symmetry equivalent reflections alone. Large improvements in behaviour of refinement, maps and statistics (R, FreeR etc.) can result from this correction.
Default: <ncyc> = 10
Default is to not use experimental sigmas in the determination. The keyword EXPE changes this to use experimental sigmas.
Default is to use all reflections in the WORKing set for scaling. The keyword FREE changes this to determine the scale from the FREE set of reflections.
NB: Before applying bulk solvent scaling and including all low resolution data, check your distribution of <F> looks sensible. This is the raw material for all overall scaling algorithms. A good way to check this is to look at a <Fsq> plot against resolution.
This should look something like this:
+ + + + + + + + + + + <10A 5A 4.5A ............
If the low resolution looks strange, it may mean your backstop was causing problems, intensities were saturated etc etc, and including such data may give unreasonable solvent scales. A sensible sort of value would be: bulk Solvent scale around -0.75 and bulk solvent B value around 200.0 if SOLVENT is not used.
We are not really sure how best to handle scaling. If you have problems please get in touch. In our experience there have been no problems with data sets with resolution 2.5Å or higher, unless there was some obvious flaw; huge ice rings or Is labelled as Fs or some such thing. But with one unusual data set which died at 2.7Å there has been a problem, which we got round by tweaking parameters, but these cases should be automatically checked.
NOTE: When doing ML refinement the scale factors are only used to calculate R values and overall B values (isotropic and anisotropic).
SCALe MLSC FIXBulk BVALue 100.0 SCVAlue -0.1
The SigmaA estimate is generally fitted to the normalised Free reflections using a 4 parameter equation of an analogous form to the bulk scaling:
SA = SA0*exp(-T0*s^2) * (1- SA1*exp(-T1*s^2))
This keyword controls the estimation of SigmaA. Subkeywords:
[Default is use the bulk solvent correction based on a constant value with the parameters of the mask VDWProb=1.4, IONProbe=0.8, RSHRink=0.8]
This keyword controls parameters for the solvent mask calculation. A constant value is assigned to the region of the unit cell not occupied by the atoms present in the input coordinate file. Its Fourier transform is used as contribution to the disordered (bulk) solvent or unmodelled part of the structure. Current version does not attempt to identify uninterpreted but ordered part of the unit cell.
Mask calculation is performed in three stages:
SOLVENT VDWProb 1.4 IONProb 0.8 RSHRink 0.8
If this keyword is active, the scale type could be set to SIMPLe. In our experience setting SCALE type to BULK and fixing BULK solvent B value to 200.0 gives "good" results:
SCALE LSSCale FIXBulk BBULk 200.0
Sometimes with high resolution data, BULK solvent B value may not be fixed.
[Default EXPE MATR 0.5]
This keyword controls the weighting of the X-ray and and geometric parts.
WEIGht MATRix 0.5
This sub-keyword allows you not to use experimental sigmas of the observations for the Xray residual. The default action is to use them.
The remaining sub-keywords control the relative weighting of the X-ray and geometry terms in the residual.
For loose restraints which is useful for high resolution data (higher than 1.5Å), this value should be increased. For example (at 1.0Å):WEIGHT MATRix 0.1
WEIGht MATRix 20
This weighting is based on the comparison between average diagonal term of X-ray and geometry "Hessians" (same as PROLSQ). Weighting equates wmat*average_diagonal_of_geometry to average_diagonal_of_Xray terms.
This keyword defines number of cycles of refinement.