Phaser 1.3 (CCP4: Supported Program)

Phaser is crystallographic software for phasing macromolecular crystal structures with maximum likelihood techniques.


Index to Documentation

1. Introduction

1.1 Keyword Index
1.2 Syntax of Documentation
1.3 Bug Reports

2. Molecular Replacement

2.1 How to Define Data
2.2 How to Define Models
2.3 How to Define Solutions
2.4 How to Control Output
2.5 How to Select Peaks
2.6 How to Run Phaser

2.6.1 Automated Molecular Replacement
2.6.2 Fast Rotation Function
2.6.3 Brute Rotation Function
2.6.4 Fast Translation Function
2.6.5 Brute Translation Function
2.6.6 Refinement and Phasing
2.6.7 Log-Likelihood Gain
2.6.8 Packing
2.6.9 Anisotropy Correction
2.6.10 Normal Mode Analysis
2.6.11 Cell Content Analysis

2.7 How to know whether Phaser has solved it
2.8 What to do in difficult cases

3. Preprocessor
4. Keywords
5. Python Scripting
6. XML
7. Version History
8. References

1. Introduction

This is the documentation for running Phaser–1.3 with keyword input. There are some minor changes to the keyword input between Phaser–1.2 and Phaser–1.3, so input scripts may need minor editing.


1.1 Keyword Index

BINS BOXScale
CELL CLMN COMPosition
EIGEn ENSEmble ENSIn
FINAl
HKLIn HKLOut
LABIn
MODE MRMAcrocycle
NORMalization NMAMethod NMAPdb
OUTLier
PACK PERMutations
RESCore RESOlution ROOT ROTAte
SAMPling SAVE SCRIpt SEARch SGALternative SHANnon SNMAcrocycle SOLUtion SOLParams SPACegroup SUITe
TARGet TITLe TOPFiles TRANslate
VERBose
XYZOut




1.2 Syntax of Documentation

KEYWord
Courier font in blue means a keyword. Only the first four letters (sometimes less) of any input keyword are required/recognized. The required characters for each keyword are given in uppercase and those not required in lowercase. Keywords are not case sensitive.


<PARAMETER>
Angle brackets mean a parameter value. Input strings are case sensitive: the case of titles and filenames is preserved.


{ KEYWord <X Y Z> }
Curly brackets mean a group of keywords/parameters must come together.


[ X | Y ]
Square brackets and line separating options means X or Y.


KEYWord <X Y Z>
Italics mean the keyword/input is optional.



1.3 Bug Reports

We apologize for the bugs. Please send bug reports to cimr-phaser@lists.cam.ac.uk or rjr27@cam.ac.uk (Randy Read).

2. Molecular Replacement

Phaser should be able to solve most structures with the Automated Molecular Replacement mode, and this is the first mode that you should try. Give Phaser your data (How to Define Data) and your models (How to Define Models), tell Phaser what to search for (use SEARch keyword), and a list of possible spacegroups (in the same pointgroup - use the SGALternative keyword). The flow diagram for the automated molecular replacement mode is shown below. If this doesn't work (see "How to know whether Phaser has solved it"), you can try selecting peaks of lower significance in the rotation function in case the real orientation was not within the selection criteria. By default peaks above 75% of the top peak are selected (see "How to Select Peaks"). See "What to do in difficult cases" for more hints and tips. If the automated molecular replacement mode doesn't work even with non-default input you need to run the modes of Phaser separately. The possibilities are endless - you can even try exhaustive searches (translations of all orientations) if you want - but experience has shown that most structures that can be solved by Phaser can be solved by relatively simple strategies.



Flow Diagram for Automated Molecular Replacement in Phaser




2.1 How to Define Data

You need to tell Phaser the name of the mtz file containing your data and the columns in the mtz file to be used using the HKLIn and LABIn keywords. Additional keywords (BINS CELL OUTLier RESOlution SPACegroup) define how the data are used.



2.2 How To Define Models

Molecular replacement models are defined with the ENSEmble keyword and the COMPosition keyword. To compute a Sigma(A) curve representing the accuracy of model structure factors as a function of resolution, Phaser needs to know the RMS coordinate error expected for the model (determined directly from RMS or indirectly from IDENtity in the ENSEmble keyword) and the fraction of the scattering power in the asymmetric unit that this model contributes (deduced from the COMPosition keywords). If fp is the fraction scattering and RMS is the rms coordinate error, then

Sigma(A) = SQRT{fp*[1-fsol*exp(-Bsol*(sin(theta)/lambda)^{^2})]} * exp{-(8 Pi^{^2}/3)*RMS^{^2}*(sin(theta)/lambda)^{^2}}
where fsol(=0.95) and Bsol(=300Å^{^2}) account for the effects of disordered solvent on the completeness of the model at low resolution.

Phaser must be given the models that it will use for molecular replacement. A molecular replacement model is constructed in one of two ways - either by making an ensemble from a set of aligned homologous structures, entered as pdb files, or by entering a model from a map, entered as structure factors in an mtz file. Each ensemble is treated as a separate type of rigid body to be placed in the molecular replacement solution. An ensemble should only be defined once, even if there are several copies of the molecule in the asymmetric unit.

If you construct a model by homology modelling, remember that the RMS error you expect is essentially the error you expect from the template structure. So specify the sequence identity of the template, not of the homology model!


Examples of building an Ensemble from Coordinates

You have one structure as a model with 44% sequence identity to the protein in the crystal.

ENSEmble mol1 PDB homology1.pdb IDENtity .44

You have three structures as models with 44%, 39% and 35% identity to the protein in the crystal.

ENSEmble mol2 PDB homology1.pdb IDENtity .44 PDB homology2.pdb IDENtity .39 PDB homology3.pdb IDENtity .35

You have an NMR Ensemble as a model. There is no need to split the coordinates in the pdb file provided that the models are separated by MODEL and ENDMDL cards. In this case the homology is not a good indication of the similarity of the structural coordinates to the target structure. You should use the RMS option; several test cases have succeeded with an RMS value of about 1.5Å.

ENSEmble mol3 PDB nmr.pdb RMS 1.5

Examples of a Map as an "Ensemble"

You have low resolution electron density of your model. This density has been cut out and converted to structure factors in a large cell.

ENSEmble mol1 HKLIn mol1.mtz F = Fmol1 P = Pmol1 EXTEnt 23 25 29 RMS 2.0 CENTre 4 3 30 PROTein MW 10241 NUCLeic MW 0

When using density as a model, it is necessary to specify both the extent (x,y,z limits) of the cut-out region of density, and the centre of this region. With coordinates, Phaser can work this out by itself. This information is needed, for instance, to decide how large rotational steps can be in the rotation search and to carry out the molecular transform interpolation correctly. In the case of electron density, the RMS value does not have the same physical meaning that it has when the model is specified by atomic coordinates, but it is used to judge how the accuracy of the calculated structure factors drops off with resolution. A suitable value for RMS can be obtained, in the case of density from an experimentally-phased map, by choosing a value that makes the SigmaA curve fall off with resolution similar to the mean figures-of-merit. In the case of density from an EM image reconstruction, the RMS value should make the SigmaA curve fall off similar to a Fourier correlation curve used to judge the resolution of the EM image.

Phaser must know what percentage of the scattering is given by each Ensemble. It can not work this out without knowing the content of the asymmetric unit. The composition of the asymmetric unit is defined either by entering the molecular weights or sequences of the components in the asymmetric unit, and giving the number of copies of each. Expert users can also enter the fraction of the scattering of each component directly, although the composition must still be entered for the absolute scale calculation.

Examples of Composition by Molecular Weight

You have one protein (with MW 21022) in the asymmetric unit

COMPosition PROTein MW 21022

You have three copies of a protein (with MW 21022) in the asymmetric unit

COMPosition PROTein MW 21022

COMPosition PROTein MW 21022

COMPosition PROTein MW 21022

Another way of entering the same thing is

COMPosition PROTein MW 21022 NUMber 3

Yet another way of entering the same thing is

COMPosition PROTein MW 63066

You have two copies of a protein (with MW 21022), two copies of a protein (with MW 9843) and RNA with (MW 32004) in the asymmetric unit

COMPosition PROTein MW 21022 NUMber 2

COMPosition PROTein MW 9843 NUMber 2

COMPosition NUCLeic MW 32004

Examples of Composition by Sequence

You have one protein (with sequence in fasta format in the file prot1.seq) in the asymmetric unit

COMPosition PROTein SEQuence prot1.seq

You have three copies of a protein (with sequence in fasta format in the file prot1.seq) in the asymmetric unit

COMPosition PROTein SEQuence prot1.seq

COMPosition PROTein SEQuence prot1.seq

COMPosition PROTein SEQuence prot1.seq

Another way of entering the same thing is

COMPosition PROTein SEQuence prot1.seq NUMber 3

Yet another way of entering the same thing is to make a sequence file with all the amino acids concatenated together (prot1.seq3)

COMPosition PROTein SEQuence prot1.seq3

You have two copies of a protein (with sequence in fasta format in the file prot1.seq), two copies of a protein (with sequence in fasta format in the file prot2.seq) and RNA with (with sequence in fasta format in the file nucl1.seq) in the asymmetric unit

COMPosition PROTein SEQuence prot1.seq NUMber 2

COMPosition PROTein SEQuence prot2.seq NUMber 2

COMPosition NUCLeic SEQuence nucl1.seq

Examples of Composition by Percentage Scattering

Each copy of Ensemble mol1 gives 22% of the scattering

COMPosition ENSEmble mol1 FRACtional 0.22

Each copy of Ensemble mol2 gives 78% of the scattering

COMPosition ENSEmble mol2 FRACtional 0.78




2.3 How To Define Solutions

You don't really need to know how to define molecular replacement solutions as Phaser writes out files ending in ".sol" and ".rlist" that contain the solution information from the job. The root of the files is given by the ROOT keyword. By default, the root filename is PHASER. These files can be read back into subsequent runs of Phaser to build up solutions containing more than one molecule in the asymmetric unit.
"PHASER.sol" files are generated by all modes, and contain the current idea of potential molecular replacement solutions.
"PHASER.rlist" files are generated by the rotation function modes, and are for performing translation functions. (They are also produced by degenerate (2D) translation functions, for performing a translation function to find the third dimension)
To include the files you should use the preprocessor command @
@ filename.sol
@ filename.rlist
However, if you want to understand "PHASER.sol" and "PHASER.rlist" files, read on…



PHASER.sol

At different stages of molecular replacement, an Ensemble will be oriented but not positioned (after the rotation search), or oriented and positioned (after the translation search), or, rarely, oriented and the position in 2 of 3 dimensions known. These three states correspond to solutions defined by the keywords SOLUtion 3DIM, SOLUtion 6DIM, and SOLUtion 5DIM. Each Ensemble in the asymmetric unit has its own SOLUtion keyword. Examples of the different types of molecular replacement solutions are:


One copy of mol1 with known orientation and position (fractional coordinates)

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

One copy of mol1 with known orientation only

SOLUtion 3DIM ENSEmble mol1 EULEr 17 20 32

One copy of mol1 with known orientation and position (fractional coordinates) and one copy of mol2 with known orientation only

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

SOLUtion 3DIM ENSEmble mol2 EULEr 5 183 230

Two copies of mol1 with known orientation and position (fractional coordinates), one copy of mol2 with known orientation and position (fractional coordinates) and one copy of mol2 with known orientation only

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

SOLUtion 6DIM ENSEmble mol1 EULEr 24 23 24 FRACtional 0.58 0.73 0.93

SOLUtion 3DIM ENSEmble mol2 EULEr 68 7 85 FRACtional 0.04 0.19 0.25

SOLUtion 3DIM ENSEmble mol2 EULEr 5 183 230


When more than one molecular replacement solution is present, the solutions are separated with the SOLUTION SET keywords.

At any given stage in the structure solution all the solutions will have the same number of ensembles oriented, and/or oriented and positioned, and the solutions will look very similar.. For example, if the rotation function and translation function for mol1 were very clear, then there will only be one type of 6DIM solution for mol1. If the rotation and translation functions for mol2 were then not clear, there will be a series of possible 6DIM solutions for mol2.


SOLUtion SET

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

SOLUtion 6DIM ENSEmble mol2 EULEr 5 183 230 FRACtional 0.71 0.54 0.81

SOLUtion SET

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

SOLUtion 6DIM ENSEmble mol2 EULEr 51 93 75 FRACtional 0.08 0.57 0.25

SOLUtion SET

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

SOLUtion 3DIM ENSEmble mol2 EULEr 5 33 21 FRACtional 0.32 0.05 0.44


Where only the coordinates in 2 dimensions (a plane through the origin) of an oriented Ensemble are determined, a solution of type 5DIM is produced. The degenerate direction is defined as the direction perpendicular to the plane in which the position is given. These solutions can be treated in exactly the same way as the 3DIM and 6DIM solutions

SOLUtion 5DIM ENSEmble mol1 EULEr 17 20 32 DEGEnerate X FRACtional 0.05 0.74



PHASER.rlist

These files define a rotation function list. The peak list is given with a series of SOLUtion TRIAl keywords.

SOLUtion TRIAl ENSEmble mol1 EULEr 17 20 32
SOLUtion TRIAl ENSEmble mol1 EULEr 67 65 51
SOLUtion TRIAl ENSEmble mol1 EULEr 67 112 81


If a partial solution is already known, then the information for the currently "known" parts of the asymmetric unit is given in the form used for the PHASER.sol file, followed by the list of trial orientations for which a translation function is to be performed.

SOLUtion SET
SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74
SOLUtion TRIAl ENSEmble mol1 EULEr 44 20 32
SOLUtion TRIAl ENSEmble mol1 EULEr 67 65 51
SOLUtion SET
SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.13 0.55 0.76
SOLUtion TRIAl ENSEmble mol1 EULEr 83 9 180
SOLUtion TRIAl ENSEmble mol1 EULEr 8 36 92
SOLUtion TRIAl ENSEmble mol1 EULEr 48 87 10


When the rlist file is generated by Phaser, an additional keyword SCORE appears on the end of the SOLUtion TRIAl lines. This is the z-score from the rotation function. It is not used by Phaser, but allows the user to keep track of the results.
SOLUtion SET
SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74
SOLUtion TRIAl ENSEmble mol1 EULEr 44 20 32 SCORe 3.4
SOLUtion TRIAl ENSEmble mol1 EULEr 67 65 51 SCORe 3.0


If a degenerate translation function is performed, then a SOLUtion TRIAl line is produced with the degenerate translation information present, ready for performing the translation function on the third dimension.
SOLUtion TRIAl ENSEmble mol1 EULEr 17 20 32 DEGEnerate X FRACtional 0.05 0.74



2.4 How to Control Output

The output of Phaser can be controlled with the following optional keywords. The ROOT keyword is not compulsory (the default root filename is "PHASER"), but should always be given, so that your jobs have separate and meaningful output filenames.

Optional Keywords
HKLOut ROOT SCRIpt SUITe TITLe TOPFiles VERBose XYZOut

Where HKLOut ON is given as an optional keyword, Phaser produces an mtz file with "SigmaA" type weighted Fourier map coefficients for producing electron density maps for rebuilding.



MTZ Column Labels		Description
FWT	PHWT	Amplitude and phase for 2m|Fobs|-D|Fcalc| exp(i alpha-calc) map
DELFWT	PHDELWT	Amplitude and phase for m|Fobs|-D|Fcalc| exp(i alpha-calc) map
FOM		m, analogous to the "Sim" weight, to estimate the reliability of alpha-calc




2.5 How to Select Peaks

The selection of peaks saved for output in the rotation and translation functions can be done in four different ways. Peaks can either be selected by "PERCent", "SIGma", "NUMber" or "ALL", illustrated below. "PERCent" means that the cutoff value is the percentage of the top peak, where the value of the top peak is defined as 100% and the value of the mean is defined as 0%. "SIGma" means that the cutoff value is the number of standard deviations (sigmas) over the mean (otherwise known as the Z-score). "NUMber" means that the cutoff value is the number of top peaks to select. "ALL" mean that all peaks are selected.

The default is selection by "PERCent" with the cutoff value set at 75%. This has the advantage that there are always peaks output. If the solution is clear, and is a long way above the mean, then only the clear solution(s) will be output, but if the distribution of peaks is rather flat, then many peaks will be output for testing in the next part of the molecular replacement procedure (e.g. many peaks selected from the rotation function for testing with a translation function). If an absolute significance test is required, then selection by "SIGma" is more appropriate, although not all searches will produce output if the cutoff value is too high (e.g. 5 sigma). If the distribution is very flat then it might be better to select by "NUMber", for example select the top 1000 rotation peaks for testing in the translation function. "ALL" is for full 6 dimensional searches, where all the solutions from the rotation function are output for testing in the translation function (although this should never be necessary; it would be much faster and probably just as likely to work if the top 1000 peaks were used in this way).

Peaks can also be clustered or not clustered prior to selection. If clustering is off, then all high peaks on the search grid are selected. If clustering is on, then points on the search grid with higher neighboring points are removed from the selection.

The selection of peaks is done in three stages for the fast rotation and fast translation searches. The first stage is the selection of peaks from the fast search that will be rescored with the full likelihood target. Rescoring with the full likelihood target may change the order of the peaks and their significance. The second stage is the selection of peaks from the rescoring to be saved and combined with other searches performed in the same phaser job. The third stage is the final selection of peaks from the merged list for output from the phaser job. The selection of peaks to go into rescoring is controlled with the RESCORE keyword, the selection of peaks saved from each separate search is controlled with the SAVE keyword, and the final selection is controlled with the FINAL keyword.

If RESCORE OFF is requested (no rescoring of the fast search peaks is performed), or if the brute rotation or translation searches are carried out, then the SAVE keyword refers to the selection of peaks from the fast search (or brute search) for merging in the final stage (the RESCORE keyword is not used for selection in this case).



2.6 How to Run Phaser

Phaser runs in different modes, which perform Phaser's different functionalities, such as rotation functions and translation functions. Some of the modes combine the functionality of other modes to allow automatic structure solution (e.g. Automated Molecular Replacement), while others are basic modes (e.g. Molecular Replacement Anisotropy Correction).

The example scripts all refer to the tutorial test case, the crystal structure of a hetero-dimer of beta-lactamase (BETA) and beta-lactamase inhibitor protein (BLIP), both with molecular replacement models from crystal structures of the individual BETA and BLIP components. The pdb and mtz files required for running this test case are distributed with Phaser.

2.6.1 Automated Molecular Replacement

This mode (MODE MR_AUTO) combines the anisotropy correction, likelihood enhanced fast rotation function, likelihood enhanced fast translation function, packing and refinement modes for multiple search models and a set of possible spacegroups to automatically solve a structure by molecular replacement. Top solutions are output to the files FILEROOT.sol, FILEROOT.#.mtz and FILEROOT.#.pdb (where "#" refers to the solution number). Many structures can be solved by running an automated molecular replacement search with defaults, giving the ensembles that you expect to be easiest to find first.

Example command script for finding BETA and BLIP. This is the minimum input, using all defaults (except the ROOT filename).
beta_blip_auto.com
phaser ‹‹ eof
TITLe beta blip automatic
MODE MR_AUTO
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
SEARch ENSEmble beta NUM 1
SEARch ENSEmble blip NUM 1
ROOT beta_blip_auto # not the default
eof


Example command script for finding BETA and BLIP. The spacegroup recorded on the mtz file is P3₂21 but the other hand is also a possibility. Both search orders (BETA first, BLIP second and BLIP first, BETA second) are tried, using the PERMutations ON keyword. We would not normally recommend using the PERMutations ON keyword for this case, as it is obvious that the larger molecule should be easier to find first.
beta_blip_auto_sg.com
phaser ‹‹ eof
TITLe beta blip automatic
MODE MR_AUTO
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
SEARch ENSEmble beta NUM 1
SEARch ENSEmble blip NUM 1
PERMutations ON # not the default
SGALternative HAND # not the default
ROOT beta_blip_auto_sg # not the default
eof


Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch

Optional Keywords
FINAl HKLOut PACK PERMutations RESCore RESOlution ROOT SAMPling SAVE SCRIpt SGALternative SOLUtion SPACegroup TITLe TOPFiles VERBose XYZOut
*BINS *BOXScale *CELL *CLMN *OUTLier *SHANnon *SOLParams *SUITe



2.6.2 Fast Rotation Function

This mode (MODE MR_FRF) combines the anisotropy correction and likelihood-enhanced fast rotation function (2), optionally rescored with the full rotation likelihood function (1), to find the orientation of a model in molecular replacement. Top rotation solutions are output to the file FILEROOT.rlist for input to a translation function. Top rotation solutions are also output to the file FILEROOT.sol.

Example command script for fast rotation function to find the orientation of BETA.
beta_frf.com
phaser ‹‹ eof
TITLe beta FRF
MODE MR_FRF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
SEARCH ENSEmble beta
ROOT beta_frf
eof


Example command script for fast rotation function to find the orientation of BLIP knowing the position and orientation of BETA, with the position and orientation of BETA input from the command line.
blip_frf_with_beta.com
phaser ‹‹ eof
TITLe blip FRF with beta rotation and translation
MODE MR_FRF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 #beta
COMPosition PROTein MW 17522 #blip
SEARch ENSEmble blip
SOLUtion 6DIM ENSEmble beta EULEr 201 41 184 FRACtional -0.49408 -0.15571 -0.28148
ROOT blip_frf_with_beta
eof


Example command script for fast rotation function to find the orientation of BLIP knowing only the orientation of BETA, with the orientation of BETA input using the output solution file from the beta_frf.com job above.
blip_frf_with_beta_rot.com
phaser ‹‹ eof
TITLe blip FRF with beta R
MODE MR_FRF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
SEARch ENSEmble blip
@beta_frf.sol # solution file output by phaser
ROOT blip_frf_with_beta_rot
eof


Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch

Optional Keywords
FINAl RESCore RESOlution ROOT SAMPling SAVE SCRIpt SOLUtion SPACegroup TITLe TOPFiles VERBose XYZOut
*BINS *BOXScale *CELL *CLMN *OUTLier *SHANnon *SOLParams *SUITe *TARGet



2.6.3 Brute Rotation Function

This mode (MODE MR_BRF) combines the anisotropy correction and brute force likelihood rotation function (1) to find the orientation of a model in molecular replacement. Top rotation solutions are output to the file FILEROOT.rlist for input to a translation function. Top rotation solutions are also output to the file FILEROOT.sol.

Example command script for brute rotation function to find the orientation of BETA
beta_brf.com
phaser ‹‹ eof
TITLe beta BRF
MODE MR_BRF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
SEARch ENSEmble beta
ROOT beta_brf
eof


Example command script for brute rotation function to find the optimal orientation of BETA in a restricted search range and on a fine grid around the position from the fast rotation search.
beta_brf_around.com
phaser ‹‹ eof
TITLe beta BRF fine sampling
MODE MR_BRF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
SEARch ENSEmble beta
ROTAte AROUnd EULEr 201 41 184 RANGE 10
SAMPling ROTation 0.5
XYZOut ON # not the default
TOPFiles 1 # not the default
ROOT beta_brf_around
eof


Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch

Optional Keywords
FINAl RESOlution ROOT Rotation SAMPling SAVE SCRIpt SOLUtion SPACegroup TITLe TOPFiles VERBose XYZOut
*BINS *BOXScale *CELL *OUTLier *SHANnon *SOLParams *SUITe



2.6.4 Fast Translation Function

This mode (MODE MR_FTF) combines the anisotropy correction and likelihood-enhanced fast translation function (3), optionally rescored by the full likelihood translation function (1), to find the position of a previously oriented model in molecular replacement. Top translation solutions are output to the file FILEROOT.sol.

Example command script for finding the position of BETA after the rotation function has been run and the results output to the file beta_frf.rlist
beta_ftf.com
phaser ‹‹ eof
TITLe beta FTF
MODE MR_FTF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
@beta_frf.rlist
ROOT beta_ftf
eof

Example command script for finding the position of BLIP after the rotation function has been run and the results output to the file blip_frf_with_beta.rlist, which has the SOLUtion 6DIM keyword input for BETA and the SOLUtion TRIAL keyword input for the orientations to try for BLIP with the translation function.
blip_ftf_with_beta.com
phaser ‹‹ eof
TITLe beta FTF
MODE MR_FTF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
@blip_frf_with_beta.rlist
ROOT blip_ftf_with_beta
eof


Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch SOLUtion

Optional Keywords
FINAl RESCore RESCore RESOlution ROOT SAMPling SAVE SCRIpt SGALternative SOLUtion SPACegroup TITLe TOPFiles VERBose XYZOut
*BINS *BOXScale *CELL *OUTLier *SHANnon *SOLParams *SUITe *TARGet



2.6.5 Brute Translation Function

This mode (MODE MR_BTF) combines the anisotropy correction and brute force likelihood translation function (1) to find the position of a previously oriented model in molecular replacement. Top translation solutions are output to the file FILEROOT.sol.

Example command script for brute Translation function to find the position of BETA after the rotation function has been run
beta_btf.com
phaser ‹‹ eof
TITLe beta BTF
MODE MR_BTF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
@beta_frf.rlist
TRANslate AROUnd FRACtional POINt -0.49408 -0.15571 -0.28148 RANGe 5
ROOT beta_btf
eof

Example command script for brute Translation function to find the position of BETA degenerate in X after the rotation function has been run
beta_btf_degen_x.com
phaser ‹‹ eof
TITLe beta degenerate X
MODE MR_BTF
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
@beta_frf.rlist
TRANslate DEGEnerate X
ROOT beta_btf_degen_x
eof

Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch SOLUtion

Optional Keywords
FINAl RESOlution ROOT SAMPling SAVE SCRIpt SGALternative SOLUtion SPACegroup TITLe TOPFiles TRANslate VERBose XYZOut
*BINS *BOXScale *CELL *COMPosition *OUTLier *SHANnon *SOLParams *SUITe



2.6.6 Refinement and Phasing

This mode (MODE MR_RNP) combines the anisotropy correction and refinement against the likelihood function (1) to optimize full or partial molecular replacement solutions and phase the data. At the end of refinement, the list of solutions is checked for duplicates, which are pruned. Refined solutions are output to the file FILEROOT.sol.
Example command script to refine a set of solutions
beta_blip_rnp.com
phaser ‹‹ eof
TITLe beta blip rigid body refinement
MODE MR_RNP
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
ROOT beta_blip_rnp # not the default
HKLOut OFF # not the default
XYZOut OFF # not the default
@beta_blip.sol
eof

Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch SOLUtion

Optional Keywords

HKLOut RESOlution ROOT SCRIpt SPACegroup TITLe TOPFiles VERBose XYZOut
*BINS *BOXScale *CELL *MRMAcrocycle *OUTLier *SHANnon *SOLParams *SUITe



2.6.7 Log-Likelihood Gain

This mode (MODE MR_LLG) combines the anisotropy correction and the likelihood function (1) to calculate the log-likelihood gain for full or partial molecular replacement solutions. Solutions are output to the file FILEROOT.sol.
Example command script to rescore the solutions using a different resolution range of data and a different spacegroup
beta_blip_llg.com
phaser ‹‹ eof
TITLe beta blip solution 6A P3121
MODE MR_LLG
HKLIn beta_blip.mtz
LABIn F=F SIGF = SIGF
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
ROOT beta_blip_llg # not the default
RESOlution 6.0
SPACegroup P 31 2 1
@beta_blip.sol
eof

Compulsory Keywords
COMPosition ENSEmble HKLIn LABIn MODE SEARch SOLUtion

Optional Keywords

HKLOut RESOlution ROOT SCRIpt SPACegroup TITLe TOPFiles VERBose XYZOut
*BINS *BOXScale *CELL *OUTLier *SHANnon *SOLParams *SUITe



2.6.8 Packing

This mode (MODE MR_PAK) determines whether molecular replacement solutions pack in the unit cell. Solutions that pack are output to the file FILEROOT.sol.

Example command script for determining whether a set of molecular replacement solutions pack in the unit cell
beta_blip_pak.com
phaser ‹‹ eof
TITLe beta blip packing check
MODE MR_PAK
HKLIn beta_blip.mtz
LABIn F=F SIGF=SIGF
ENSEmble beta PDB beta.pdb IDENtity 100
ENSEmble blip PDB blip.pdb IDENtity 100
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
ROOT beta_blip_pak # not the default
PACK 1 # not the default
@beta_blip.sol
eof

Compulsory Keywords
ENSEmble HKLIn LABIn MODE SEARch SOLUtion

Optional Keywords

PACK ROOT SCRIpt SPACegroup TITLe VERBose XYZOut
*CELL *SUITe



2.6.9 Anisotropy Correction

This mode (MODE MR_ANO) corrects the experimental data for anisotropy. Data (amplitude and associated sigma) are corrected for anisotropy and output to FILEROOT.mtz with column label set to the input column label with the addition of _ISO.

Example command script to phase a molecular replacement solution only
beta_blip_ano.com
phaser ‹‹ eof
TITLe beta blip data correction
MODE MR_ANO
HKLIn beta_blip.mtz
LABIn F=Fobs SIGF=Sigma
ROOT beta_blip_ano # not the default
eof

Compulsory Keywords
HKLIn LABIn MODE

Optional Keywords
ROOT SCRIpt SPACegroup TITLe VERBose
*CELL *SNMAcrocycle *SUITe



2.6.10 Normal Mode Analysis

This mode (MODE MR_NMA) writes out pdb files that have been perturbed along normal modes, in a procedure similar to that described by Suhre & Sanejouand (Acta Cryst. D60, 796-799, 2004). Each run of the program writes out a matrix FILEROOT.mat that contains the eigenvectors and eigenvalues of the atomic Hessian, and can be read into subsequent runs of the same job, to speed up the analysis.

Do normal mode analysis only, write out eigenfile but not coordinates
beta_nma.com
phaser ‹‹ eof
TITLe beta normal mode analysis
MODE MR_NMA
ENSEmble beta PDB beta.pdb IDENtity 100
XYZOut OFF
ROOT beta_nma # not the default
eof

Write out pdb files perturbed in 0.5 angstrom rms intervals along modes 7 and 8 (and combinations of 7 and 8)
beta_nma_pdb.com
phaser ‹‹ eof
TITLe beta normal mode analysis pdb file generation
MODE MR_NMA
ENSEmble beta PDB beta.pdb IDENtity 100
ROOT beta_nma_pdb # not the default
EIGEn beta_nma.mat
NMAPdb MODE 7 MODE 8 RMS 0.5
eof

Compulsory Keywords
ENSEmble MODE

Optional Keywords
NMAMethod ROOT SCRIpt TITLe VERBose XYZOut EIGEn NMAPdb
*SUITe



2.6.11 Cell Content Analysis

This mode (MODE MR_CCA) determines the composition of the crystals using the "new" Matthews coefficients of Kantardjieff & Rupp (2003) "Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA and protein-nucleic acid complex crystals". Protein Science 12:1865-1871. The molecular weight of ONE complex or assembly to be packed into the asymmetric unit is given with the COMPosition keyword, and the possible Z values (number of copies of the complex or assembly) that will fit in the asymmetric unit and the relative frequency of their corresponding VM values is reported. RESOlution should be set to the maximum resolution that has been observed for the crystal.

Do cell content analysis
beta_cca.com
phaser ‹‹ eof
TITLe beta-blip cell content analysis
MODE MR_CCA
COMPosition PROTein MW 28853 NUM 1 #beta
COMPosition PROTein MW 17522 NUM 1 #blip
RESO 3.0
ROOT beta_blip_cca # not the default
eof

Compulsory Keywords
COMPosition MODE

Optional Keywords
SEARch RESOlution ROOT TITLe VERBose
*SUITe



2.7. How to know whether Phaser has solved it

By default, Phaser selects solutions over 75% of the the difference between the top solution and the mean. Ideally, only the number of solutions you are expecting should be selected by this criterion, but if the signal-to-noise of your search is low, there will be noise peaks in this selection also. For a translation function the correct solution will generally have a Z-score (number of standard deviations above the mean value) over 5 and be well separated from the rest of the solutions. For a rotation function, the correct solution may be in the list with a Z-score under 4, and will not be found until a translation function is performed and picks out the correct solution.

Of course, there will always be exceptions! Note, in particular, that in the presence of translational NCS, pairs of similarly-oriented molecules separated by the correct translation vector will give large Z-scores, even if they are incorrect, because they explain the systematic variation in intensities caused by the translational NCS.



Z-score	Have I solved it?
less than 5	no
5 - 6	unlikely
6 - 7	possibly
7 - 8	probably
more than 8	definitely




You should always at least glance through the summary log file. One thing to look for, in particular, is whether any translation solutions with a clear signal-to-noise have been rejected by the packing step, especially with a small number of clashes. Such a solution may be correct, and the clashes may arise only because of differences in small surface loops. If this happens, repeat the run allowing a suitable number of clashes with the PACK keyword.

2.8. What to do in difficult cases

Not every structure can be solved by molecular replacement, but the right strategy can push the limits. What to do when the default jobs fail depends on why your structure is difficult.

Flexible structure

The relative orientations of the domains may be different in your crystal than in the model. If that may be the case, break the model into separate PDB files containing rigid-body units, enter these as separate ensembles, and search for them separately. If you find a convincing solution for one domain, but fail to find a solution for the next domain, you can take advantage of the knowledge that its orientation is likely to be similar to that of the first domain. The ROTAte AROUnd option of the brute rotation search can be used to restrict the search to orientations within, say, 30 degrees of that of the known domain. Allow for close approach of the domains by increasing the allowed clashes with the PACK keyword by, say, 1 for each domain break that you introduce.

Alternatively, you could try generating a series of models perturbed by normal modes, with the NMAPdb keyword. One of these may duplicate the hinge motion and provide a good single model.

Poor or incomplete model

Signal-to-noise is reduced by coordinate errors or incompleteness of the model. Since the rotation search has lower signal to begin with than the translation search, it is usually more severely affected. For this reason, it can be very useful to use the subsequent translation search as a way to choose among many (say 1000) orientations. Try increasing the number of clustered orientations in an AUTO job using the keyword FINAl, e.g. FINAl ROT SELEct PERCent 65. If that fails, try turning off the clustering feature in the save step (SAVE ROT CLUSter OFF), because the correct orientation may sit on the shoulder of a peak in the rotation function.

As shown convincingly by Schwarzenbacher et al. (Schwarzenbacher, Godzik, Grzechnik & Jaroszewski, Acta Cryst. D60, 1229-1236, 2004), judicious editing can make a significant difference in the quality of a distant model. In a number of tests with their data on models below 30% sequence identity, we have found that Phaser works best with a "mixed model" (non-identical sidechains longer than Ser replaced by Ser). In agreement with their results, the best models are generally derived using more sophisticated alignment protocols, such as their FFAS protocol.

High degree of non-crystallographic symmetry

If there are clear peaks in the self-rotation function, you can expect orientations to be related by this known NCS. Methods to automatically use such information will be implemented in a future version of Phaser. In the meantime, you can work out for yourself the orientations that would be consistent with NCS and use the ROTAte AROUnd option to sample similar orientations. Alternatively, you may have an oligomeric model and expect similar NCS in the crystal. First search with the oligomeric model; if this fails, search with a monomer. If that succeeds, you can again use the ROTAte AROUnd option to force a subsequent monomer to adopt an orientation similar to the one you expect.

Pseudo-translational non-crystallographic symmetry

It is frequently the case that crystallographic and non-crystallographic rotational symmetry axes are parallel. The combination generates translational NCS, in which more than one unique copy of the molecule is found in the same orientation in the crystal. This can be recognized by the presence of large non-origin peaks in the native Patterson map. If one copy of the search model can be found, then the translational NCS tells you where to place another copy. Unfortunately, the presence of translational NCS can make it difficult to solve a structure using Phaser, because the current likelihood targets do not account for the statistical effects of NCS.

3. Preprocessor

Preprocessor commands (@ # & END GO RUN START STOP QUIT EXIT KILL) may be used in the keyword input to incorporate files, add comments or allow line continuation.
• To include a file in the input stream use @<filename>
• All characters on a line after a hash (#) character are ignored
• Line continuation with the ampersand (&) character
• END GO RUN START STOP QUIT EXIT KILL or "eof" from a command file ends the input and starts Phaser

4. Keywords

Phaser can be controlled using keyword input. Not all keywords are relevant for all modes of operation (the list of relevant keywords for each mode is given with each mode above). Some keywords are only for single use, others have meaning when used more than once. The input values of many parameters are constrained to physically meaningful values. All non-compulsory parameters have defaults.



Asterisk (*)		Keywords are for "expert" use only, or use in development.
Constraints	X=integer(+)	If X is a positive integer, stored value = X, else error
Constraints	X=%	If 1<X<=100, stored value = X, else if 0<X<1, stored value = X*100, else error
Default		Some default values are constants, others are set by Phaser after it has analyzed the input data.
Single Use		Keyword only applicable once. If used multiple times, the last value input will be used.
Multiple Use		Keyword is meaningful when entered multiple times. The order may or may not be important.







*BINS {MINimum <L>} {MAXimum <H>} {NUMber <N>} {WIDTh <W>} {CUBIc <A B C>}

The binning of the data. L = minimum number of bins, H = maximum number of bins, N = number of bins, W = width of the bins in number of reflections, A B C are the coefficients for the binning function A(S*S*S)+B(S*S)+CS where S = (1/resolution). If N is given then the values of L and H are ignored.
Single Use
Constraints L,H,N,W=integer(+) CUBIc restricted to monotonically increasing: Either (a) AC >0,BC >0,C >0 or (b) A=B=0 or (c) A=0 or (d) B=0
Default BINS MINimum 6 MAXimum 50 WIDTh 1000 CUBIc 0 1 0




*BOXScale <BOXSCALE>

Scale for box for calculating structure factors. The ensembles are put in a box equal to (extent of molecule)*BOXSCALE. This BOXSCALE applies to all ensembles, except those for which BOXSCALE has been set individually using the ENSEmble keyword.
Single Use
Constraints BOXSCALE >2.4
Default BOXScale 4




*CELL <A B C ALPHA BETA GAMMA>

Unit cell dimensions
Single Use
Constraints A>0,B>0,C>0,ALPHA>0,BETA>0,GAMMA>0
Default Cell read from MTZ file




*CLMN {SPHEre <SPHERE>} {LMINimum <LMIN>} {LMAXimum <LMAX>}

The radii or L values for the decomposition of the Patterson in Ångstroms.
Single Use
Constraints SPHERE>5,LMIN>0,LMAX>LMIN
Default CLMN SPHEre <2*geometric mean radius of Ensemble> LMIN 2




COMPosition MATThews { BEST <NUM> | ALL }

Composition of the crystals estimated using the "new" Matthews coefficients of Kantardjieff & Rupp (2003) "Matthews coefficient probabilities: Improved estimates for unit cell contents of proteins, DNA and protein-nucleic acid complex crystals". Protein Science 12:1865-1871
Single Use
Default Composition determined by Matthews coefficient

COMPosition [PROTein | NUCLeic] [MW <MW> | SEQuence <FILE>] NUMber <NUM>

Composition of the crystals. The number of copies NUMber of molecular weight MW or SEQuence given in fasta format in FILE of protein or nucleic acid in the asymmetric unit.
Multiple Use
Constraints MW>0, NUMber=integer(+)
Default None for MW, compulsory when required. NUMber defaults to 1.

COMPosition ATOM <ATOMTYPE> <NUM>

NUM atoms of ATOMTYPE are added to the composition.
Multiple Use

COMPosition SCAttering <SCATTERING>

Add scattering directly.
Multiple Use

COMPosition NUM <NUM>

If the composition given by other COMP PROT, COMP NUCL and COMP ATOM cards represents a complex in the asymmttric unit then COMP NUM can be used to give how many times this complex is present in the asymmtric unit.
Single Use

COMPosition ENSEmble <MODLID> FRACtional <FRAC>

Alternative way of defining composition. Fraction scattering is entered explicitly for each ENSEmble .
Multiple Use
Constraints 0<FRAC<=1
Default None, compulsory when required

 




EIGEn [ {READ <EIGENFILE>} | {WRITe [ON|OFF]} ]

READ or WRITe a file containing the eigenvectors and eigenvalues. If reading, the eigenvalues and eigenvectors of the atomic Hessian are read from the file generated by a previous run, rather than calculated. This option must be used with CAUTION: the job that generated the eigenfile and the job reading the eigenfile must have identical (or default) input for keyword NMAMethod. Use WRITe to control whether or not the eigenfile is written when not using the READ mode.
Single Use
Default EIGEN WRITE ON




ENSEmble <MODLID> PDB <PDBFILE> [RMS | IDENtity] <NUM> {PDB <PDBFILE> [RMS | IDENtity] <NUM>}…

The names of the PDB files used to build the ENSEmble , and either the expected RMS deviation of the coordinates to the "real" structure or the percent sequence identity with the real sequence.
Multiple Use
Constraints NUM=% if IDENtity
Default None, compulsory when required

ENSEmble <MODLID> BOXScale <BOXSCALE>

Boxscale for building ensemble.
Multiple Use
Constraints BOXSCALE > 0
Default BOXScale 4

ENSEmble <MODLID> HKLIn <MTZFILE> F = <F> PHI = <P> EXTEnt <EX EY EZ> RMS <RMS> CENTre <CX CY CZ> PROTein MW <PMW> NUCLeic MW <NMW>

An ENSEmble defined from a map (via an mtz file). The molecular weight of the object the map represents is required for scaling. The effective RMS coordinate error is needed to judge how the map accuracy falls off with resolution.. The extent is needed to determine reasonable rotation steps, and the centre is needed to carry out a proper interpolation of the molecular transform. The extent and the centre are both given in Angstroms.
Multiple Use
Default None, compulsory when required

*ENSEmble <MODLID> BINS {MINimum <L>} {MAXimum <H>} {NUMber <N>} {WIDTh <W>} {CUBIc <A B C>}

Bins for the MODLID
Multiple Use
Constraints L,H,N,W = integer(+) CUBIc restricted to monotonically increasing: Either (a) AC >0,BC >0,C >0 or (b) A=B=0 or (c) A=0 or (d) B=0
Default ENSEmble <MODLID> BINS MINimum 6 MAXimum 200 WIDTh 1000 CUBIc 0 1 0




*ENSIn <MODLID> HKLIn <MTZFILE> SCATtering <SCAT> EXTEnt <EX EY EZ> PR <P1 P2 P3 P4 P5 P6 P7 P8 P9> PT <TX TY TZ>


This option can be used (but rarely is) to read back a molecular transform computed in an earlier Phaser job run in the MR_ENS mode.
Single Use
Constraints S>0,N=integer(+),P=%
Default FINAl SELEct PERCent 75




FINAl [ROT | TRA] SELEct [{SIGma <S>} | {NUMber <N>} | {PERCent <P>} | ALL]


Final selection criteria for peaks. If no criteria are given for saving or rescoring steps, then the criteria given for final selection is used as the criteria for the saving and rescoring steps also.
Single Use
Constraints S>0,N=integer(+),P=%
Default FINAl SELEct PERCent 75




HKLIn <FILENAME>

The mtz file containing the data
Single Use
Default None, compulsory when required




HKLOut [ON|OFF]

Flags for output of an mtz file containing the phasing information
Single Use
Default HKLOut ON




LABIn F = <F> SIGF = <SIGF>

Columns in mtz file. F must be given. SIGF should be given but is optional.
Single Use
Default None, compulsory when required




MODE [MR_AUTO | MR_FRF | MR_FTF | MR_BRF | MR_BTF | MR_RNP | MR_LLG | MR_PAK | MR_ANO]

The mode of operation of Phaser
Single Use
Default None, compulsory




NMAMethod {RTB {NRESidues <NRES>} {MAXBlocks <MAXBLOCKS>} | CA | ALL } {RADIus <RADIUS>} {FORCe <FORCE>}

Input for the normal mode analysis. Writes out pdb files perturbed along normal mode(s). {RTB|CA|ALL} define the atoms used for the analysis. RTB uses the rotation-translation block method, CA uses C-alpha atoms only to determine the modes, and ALL uses all atoms to determine the modes (only for use on very small molecules, less than 250 atoms). For the RTB analysis, by default NRES is calculated so that it is as high as it can be without reaching MAXBlocks. The interaction radius used for the calculations and the force constant can also be altered.
Single Use
Default RTB MAXBlocks 250 RADIus 5.0 FORCe 1.0




NMAPdb {MODE <M1> {MODE <M2>…}} {RMS <RMS>} {CLASh <CLASH>} {STREtch <STRETCH>} {MAXRms <MAXRMS>} {FORWard | BACKward | TOFRo} {DQ <DQ1> {DQ <DQ2>…}} {COMBination [ON|OFF]}

MODE is the mode along which to perturb the structure. If multiple modes are entered, the structure is perturbed along all the modes AND combinations of the modes given. There is no limit on the number of modes that can be entered, but the number of pdb files explodes combinatorially. RMS is the increment in rms angstroms between pdb files to be written. The structure will be perturbed along each mode until either the C-alpha atoms clash with (come within CLASH angstroms of) other C-alpha atoms, the distances between C-alpha atoms STREtch too far (note that normal modes do not preserve the geometry) or the MAXRMS deviation has been reached. The structure is perturbed either forwards or backwards or to-and-fro (FORWard|BACKward|TOFRo) along the eigenvectors of the modes specified. Alternatively, the DQ factors (as used by the Elnemo server (K. Suhre & Y-H. Sanejouand, NAR 2004 vol 32) ) by which to perturb the atoms along the eigenvectors can be entered directly. The keyword COMBination controls whether or not only those structures that are a combination of all modes are written out (i.e. no displacements of zero along any of the modes).
Single Use
Default MODE 7 RMS 1.0 TOFRo STREtch 5.0 CLASh 2.0 MAXRms 50.0 COMBination OFF




*MRMAcrocycle ROTation [ON|OFF] TRAnslation [ON|OFF] NCYCle <NCYC> MINImizer <MINIMIZER>

Molecular replacement refinement macrocycle. The macrocycles are performed in the order that they are entered
Multiple Use
Default MRMAcrocycle ROTation ON TRAnslation ON NCYCle 20 MINImizer BFGS

 

*NORMalization {BINS <B1 B2 B3 …>} {ANISotropic <HH KK LL HK HL KL>} {ISOB <ISOB>} {SOLK <SOLK>} {SOLB <SOLB>} FIXB FIXA FIXS FIXK

Normalization parameters for the data. Normalization factor for reflection hkl in bin i is given by SigmaN = Bi*(1-SOLK*exp(-SOLB))* exp(-(HH*h*h + KK*k*k + LL*l*l + HK*h*k + HL*h*l + KL*k*l))
Single Use
Default Calculated by Phaser




*OUTLier [{ON <OUTLIER_PROB>} | OFF]

Control of the large unlikely E-value rejection. Outliers with a probability less than OUTLIER_PROB are rejected.
Single Use
Default OUTLier ON 0.000001




PACK <ALLOWED_CLASHES>

NUMber of C-alpha atoms that can clash within 2A.
Single Use
Default PACK 0




PERMutations [ON|OFF]


Toggle for whether the order of the search set is to be permuted.
Single Use
Default PERMutations OFF




*REFLection <H K L FMEAN SIGFMEAN>


Reflection data input manually.
Multiple Use
Default None





RESCore [ROTation | TRAnslation] [ON|OFF]


Toggle for rescoring of fast search peaks
Single Use
Default RESCore ON

RESCore [ROTation | TRAnslation] SELEct [{SIGma <S>} | {NUMber <N>} | {PERCent <P>} | ALL]


Selection criteria for peaks to be rescored.
Single Use
Constraints S>0,N=integer(+),P=%
Default RESCore SELEct PERCent 67.5

RESCore [ROTation | TRAnslation] CLUSter [ON|OFF] {DUMP <NDUMP>} {LOG [ON|OFF]}

CLUSter ON or OFF selects whether raw or clustered peaks are to be used in the rescoring. If clustered peaks are used, then NDUMP raw peaks will be dumped to the output. If clustered peaks are not used, then you may still perform the clustering and log the results to the log file (this may be time consuming if a large number of peaks are selected for clustering).
Single Use
Constraints NDUMP>0

Default RESCore CLUSter OFF LOG ON




RESOlution <HIRES> <LORES>

Resolution range in Angstroms. If only one limit is given, it is the high resolution limit; otherwise the limits can be in either order.
Single Use
Constraints HIRES>0,LORES >0
Default Resolution range set to accommodate all input reflections




ROOT <FILEROOT>

Root filename for output files (e.g. FILEROOT.log)
Single Use
Default ROOT PHASER




ROTAte FULL

Sample all unique angles

ROTAte AROUnd EULEr <A B G> RANGe <RANGE>

Restrict the search to the region of +/- RANGE degrees around orientation <A B G>
Single Use
Constraints RANGE>0
Default ROTAte FULL




SAMPling {ROTation <ROTSAMP>} {TRAnslation <TRASAMP>}

Sampling of search given in degrees for a rotation search and Angstroms for a translation search.
Single Use
Constraints ROTSAMP>0, TRASAMP>0
Default SAMPling ROTSAMP <2*atan(dmin/(4*geometric mean radius))> TRASAMP <dmin/5> for brute force search, <dmin/3> for fast translation search





SAVE [ROTation | TRAnslation] SELEct [{SIGma <S>} | {NUMber <N>} | {PERCent <P>} | ALL]

Peaks satisfying selection criteria are saved. If no criteria are given for the rescoring step, then the criteria given for saving is used as the criteria for the rescoring step also.
Single Use
Constraints S>0,N=integer(+),P=%
Default SAVE SELEct PERCent 75

SAVE [ROTation | TRAnslation] CLUSter [ON|OFF] {DUMP <NDUMP>} {LOG [ON|OFF]}

CLUSter ON selects clustered peaks for saving. If clustered peaks are used, then NDUMP raw peaks will be dumped to the output. CLUSter OFF selects raw peaks for saving. If clustered peaks are not used, then you may still perform the clustering and log the results to the log file (this may be time consuming).
Single Use
Constraints NDUMP>0
Default SAVE CLUSter ON DUMP 20




*SCRIpt [ON|OFF]

Write Phaser script file
Single Use
Default SCRIpt ON




SEARch ENSEmble <MODLID> {OR ENSEmble <MODLID>}… {NUMber <NUM>}

The ENSEmble to be searched for in a rotation search or an automatic search. When multiple ensembles are given using the OR keyword, the search is performed for each ENSEmble in turn. The final results are the best of all the searches (controlled with the FINAL keyword). When the keyword is entered multiple times in the MR mode, each SEARCH keyword refers to a new component of the structure. If the component is present multiple times the sub-keyword NUMber can be used (rather than entering the same SEARCH keyword NUMber times). If the MR mode is being used with a fixed partial solution, only enter SEARCH keywords (or associated NUMbers) for the components that remain to be found.
Multiple Use
Constraints ENSEmble MODLID must be defined
Default None, compulsory when required

SEARch MATThews [ON | OFF]

The number of copies of the search components to search for is determined by the Matthews coefficient. The stoichiometry of the search is given using the NUM keyword above.
Single Use
Default SEARCH MATThews BEST 1




SGALternative [ALL | HAND | {TEST <SG>}]

Alternative space groups to test in the translation function. All tests all possible space groups, hand tests the given spacegroup and its enantiomorph and <SG> tests the give space group.
Multiple Use
Default None




*SHANnon <SHARAT>

Shannon sampling given by (2*SHARAT) for the Ensemble maps. Increase SHARAT to 2 to sharpen the sampling.
Single Use
Constraints SHARAT>1.1
Default SHANnon 1.5




*SNMAcrocycle ANISotropic [ON|OFF] BINS [ON|OFF] SOLK [ON|OFF] SOLB [ON|OFF] NCYCle <NCYC> MINImizer <MINIMIZER>

Macrocycles for the refinement of SigmaN in the anisotropy correction
Multiple Use
Constraints NCYC=integer(+)
Default SNMAcrocycle ANISotropic ON BINS ON SOLK OFF SOLB OFF NCYCle 50 MINImizer BFGS

*SNMAcrocycle OFF

Turns off the anisotropy correction





SOLUtion SET <ANNOTATION>

Start new set of solutions
Multiple Use

SOLUtion 3DIM ENSEmble <MODLID> EULEr <A B G> FIXR

Rotation only solution. Use this keyword if only the orientation in 3 dimensions is known. This keyword is repeated for each case. A B G are the Euler angles in degrees.
Multiple Use

SOLUtion 5DIM ENSEmble <MODLID> EULEr <A B G> DEGEnerate [X|Y|Z] FRACtional <U V> FIXR FIXT

Use this keyword if the orientation in 3 dimensions and and the position of the MODLID in only 2 dimensions is known. This keyword is repeated for each case. A B G are the Euler angles in degrees. The keywords [X|Y|Z]specify the degenerate direction and U V are the translation in the other two directions.
Multiple Use

SOLUtion 6DIM ENSEmble <MODLID> EULEr <A B G> [ORTHogonal | FRACtional] <X Y Z> FIXR FIXT

This keyword is repeated for each known position and orientation of a ENSEmble ID. A B G are the Euler angles and X Y Z are the translation.
Multiple Use

SOLUtion TRIAl ENSEmble <MODLID> EULEr <A B G> {DEGEnerate [X|Y|Z] FRACtional <U V> } {SCORe <score>}

Rotation List for translation function
Multiple Use
Default none, compulsory when required

 


*SOLParams <FSOL> <BSOL>

Optionally change solvent parameters for Sigma(A) curves from the default values. The results are not terribly sensitive to these parameters, which affect only lower resolution data. FSOL and BSOL can be given in either order, the lower number being taken as FSOL.
Single Use
Constraints 0<FSOL<1 and BSOL>0
Default FSOL=0.95 BSOL=300




SPACegroup {HALL}