MOLREP (CCP4: Supported Program)¶
NAME¶
molrep - automated program for molecular replacement
SYNOPSIS¶
DESCRIPTION¶
Version 11.0 /22.07.2010/¶
Installation and Downloads (source code, binaries,tutorials)
MOLREP version to read MTZ file
Molecular replacement method. Some theory.
How to redirect output and scratch files
Convention for rotation and coord. system
FEATURES¶
standard molecular replacement method by cross rotation function (RF), full-symmetry translation function (TF), packing function (PF) also Self Rotation Function with PostScript plots Spherically averaged phased translation function (SAPTF) Phased Rotation function(PRF) Phased Translation function (PTF) Locked cross rotation function (LRF)
allows input of a priori knowledge of similarity of the model.
scaling by Patterson origin peak soft low resolution cut-off anisotropic correction and scaling can use modified stucture factors instead of Fobs for RF
can use second fixed model
can check and manage pseudo-translation
can use MTZ file
can just rotate and position the model and compute R-factor, CC
search model in electron density or EM map
can choose from symmetry-related models closest to which was found before
can improve the model before to use model correction by sequence alignment
can use NMR model
can use EM or electron density map as model or use it instead of Fobs for searching a atomic model in EM map
search model orientation in electron density map for particular position by phased RF
find HA positions by MR solution
REFERENCES¶
Author: A.A.Vagin email: alexei@ysbl.york.ac.uk References: A.A.Vagin, New translation and packing functions., Newsletter on protein crystallography., Daresbury Laboratory, (1989) 24, pp 117-121. Alexei Vagin and Alexei Teplyakov. An approach to multi-copy search in molecular replacement., Acta Cryst.D,(2000) 56, pp 1622-1624 A.A.Vagin and M.N.Isupov Spherically averaged phased translation function and its application to the search for molecules and fragments in electron-density maps Acta Cryst.D,(2001) 57, pp 1451-1456 main: A.Vagin,A.Teplyakov, MOLREP: an automated program for molecular replacement., J. Appl. Cryst. (1997) 30, 1022-1025.
INSTALLATION¶
Copy file molrep.tar.gz
and uncompress it (`gunzip molrep.tar.gz’)
After untaring `molrep.tar’ (command: tar xvf molrep.tar) you will get a molrep directory, with src, doc, data, molrep_check and bin subdirectories and README file. To build the executable, go to src.
- 1. setenv BLANC_FORT
define compiler with options,for example: for linux and mac: setenv BLANC_FORT “f90 -fno-globals -fno-automatic -O1 -w” for linux intel compiler: setenv BLANC_FORT “ifort -static -O1” for mac ibm compiler: setenv BLANC_FORT “ xlf -qextname -qarch=auto -qtune=auto -qstrict -O3” else setenv BLANC_FORT “f90 -O1”
- 2. csh molrep.setup
the executable (molrep) will finish up in the bin directory; providing the full pathname (…/molrep/bin/molrep) one can execute it from anywhere without having to define an environmental variable. CCP4 version (which can read MTZ file ) will be prepared automaticly if ccp4 is installed
Also you can download binaries (executable files):
Tutorials:
also you can use Testing_program_MOLREP as tutorial
New style to use¶
You can use this version as previous one:
1. by command (batch) file
2. interactively
3. by ccp4i
New style to use:
You can use program by command string with options:
molrep -f file_sf_or_map -m model_crd_or_map
-mx fixed model -m2 model_2
-po path_out -ps path_scrath
-s file_sequence -s2 file_seq_for_m2
-k file_keywords -doc y_or_a_or_n
-h -i -r
h = only keyword and mtz label information, clean
i = interactive mode
r = rest some special files
file_keywords = simple text file with keywords
(one line - one keyword)
Examples:
Without any keywords:
Usual MR: RF + TF
molrep -f file.mtz -m model.pdb
Usual MR with fixed model
molrep -f file.mtz -m model.pdb -mx mfix.pdb
Usual MR with sequence and redirect output and scratch files
molrep -f file.mtz -m model.pdb -s file_seq -po out/ -ps scr/
Self rotation funtion
molrep -f file.mtz
multi-copy search, one model (DYAD M)
molrep -f file.mtz -m model.pdb -m2 model.pdb
multi-copy search, two different models (DYAD M)
molrep -f file.mtz -m model1.pdb -m2 model2.pdb
Fitting two atomic models
molrep -m model1.pdb -mx model2.pdb
Rigid body refinement
molrep -f file.mtz -mx model.pdb
Get information about keywords and mtz labels, and clean
molrep -h -f file.mtz
If you like to play with keywords:
Using keywords from file
molrep -f fobs.pdb -m model.pdb -k file_keywords
Using keywords interactivly
molrep -f file.mtz -m model.pdb -i "CR"
sim .4 "CR"
sg all "CR"
"CR"
Script example:
--------------------------------
molrep -f fobs.mtz -m model.pdb -i <<stop
fun t
file_t tab
stick n
stop
--------------------------------
News and New keywords¶
Use “molrep -h” to get short manual of MOLREP.
You can stop program safely if you create in current directory or in PATH_OUT (if option -po is used) file:
molrep_stop_signal.xml
(contents does not matter)
DYAD < M >¶
new value M means “multi-monomer search”, new algorithm of Multi-copy search . Instead to search two copies and then next one program constructs maximal possible complexes of monomers (3mres,4mers,5mers,…) and checks its by TF. |
SCORE < Y | N | C >¶
N means do not stop if contrast is good and do not assess solution. Scoring system is working well if expected number of models and proper symmetry of model are correct.Proper symmetry of model program defines by model Self Rotation Funtion when Cross Rotation function is computed. If you like or if you use keyword FUN = T you can define Proper symmetry of model by keyword NCSM. |
|
C use Correlation Coeffitient instead of score and do not stop. score is product Correlation Coeffitient, value of Packing function and maximal value of Packing index. contrast means: >3.0 - definitly solution <3.0 and > 2.0 - solution <2.0 and > 1.5 - maybe solution <1.5 and > 1.3 - maybe not solution, but program accepts it <1.3 - probably not solution. |
NCSM <auto>¶
PST <A>¶
new value A means to check all pseudo-translation vectors automatically (default now). |
DIFF <M>¶
new value M means to remove fixed model from map (Fobs,PHobs) by mask for RF or PRF. |
SG <name>¶
space group name. You can use this keyword instead NOSG ALL means to check all possible space groups automatically. |
RESMIN <auto>¶
SEQ < D | Y | N >¶
D |
default: to use identity as SIM and to use corrected model only if identity > 20% |
N |
means to use identity as SIM only (without model correction) |
Y |
means to use identity as SIM and to use corrected model |
SURF <C >¶
SURFX < Y | C | N >¶
Y is default. SURF for fixed model (no shift to origin) |
SELF < N | A >¶
A |
after RF program computes Self_RF and use from RF only related peaks for TF. |
INPUT/OUTPUT FILES¶
Input file formats
Format of input file of models
for atomic model - PDB or CIF or BLANC.
for EM or electron density map - CCP4 or BLANC. CCP4 file must have extension “ccp4” or “map”
Input file of structure factors and phases.
Input formatted file of structure factors (CIF). This file must be CIF file which contains indices, structure factors (and phases if you need them): h,k,l,|F|,sig(F),Phi or h,k,l,|F|,sig(F),Phi,Fom
Input PDB file of structure factors. This file contains indices and structure factors or intensities. (also simple formatted file with h,k,l,|F|,sig(F) or *h,k,l,|F| * and without titles is acceptable)
Alternative input file of structure factors or phases is unformatted file of BLANC suite.
MTZ file, which must have extension “mtz”.
EM or electron density file (CCP4 file must have extension “ccp4” or “map”). This file will be converted to files of !F! and phases.
You can find some examples in Input file examples.
Space group and unit cell parameters of the unknown structure will be taken from the file of structure factors. You can change the space group of the structure factor file by using keywords NOSG or SG .
Output files¶
- molrep.crd
new coordinates of model (plus model_2) corresponding to the best solution of Cross Rotation and Translation Function.(BLANC format)
- molrep.pdb
new PDB coordinate file with molrep solution (this file will be created if you start from PDB file).
molrep_fcalc.dat molrep_phcalc.dat
!F! and phases of molrep solution (plus from model_2). These files will be created if your model is EM or density. (BLANC format)
- molrep_fcalc.cif
formatted CIFile of molrep solution (plus from model_2) with Fobs, Fcalc, Phcalc (this files will be created if your model is EM or density in CCP4 format).
- molrep.doc
full protocol of calculation (text format).
- molrep.btc
command (batch) file. You can repeat calculation using this file: cp molrep.btc bat and sh bat
- molrep_rf.tab
List of peaks of rotation function, created as soon as the program calculates a Rotation Function. molrep_rf.tab is default name, you can use another name using keyword FILE_T. You can edit this file and use it for subsequent calculations without computing the rotation function again (keyword FUN=T). The program then reads (in free format!): “Sol_”, peak number and Polar angles (theta,phi,chi), e.g.:
"Sol_ 23 10.0 22.2 40.0"In Phased Rotation Function calculations, the program reads “Sol_”, peak number, Polar angles and shift (sx,sy,sz), e.g.:
"Sol_ 23 10.0 22.2 40.0 .564 .443 .032"In Rotation and Position the model (keyword FUN=S), the program reads “Sol_”, peak number,Polar angles and shift (sx,sy,sz) e.g.:
"Sol_ 23 10.0 22.2 40.0 .564 .443 .032"If you like to use Eiler angles use “Sol_A” instead “Sol_”
In ‘Search model orientation by PRF’ (keyword PRF=P), the program reads “Sol_”, peak number,Polar angles and shift (sx,sy,sz) e.g.:
"Sol_ 23 10.0 22.2 40.0 .564 .443 .032"But program will use only the shift (sx,sy,sz).
- molrep_srf.tab
List of peaks of Self Rotation Function. molrep_srf.tab is default name, you can use another name using keyword FILE_TSRF.
- molrep_rf.ps
PostScript file of Self Rotation Function
Some output files will not be deleted if option “-r” in command string was used. They have the internal format of the BLANC program suite and can be used by programs of this suite.
- crossrot_alo.dat
spherical coefficients of F_observed
- crossrot_alm.dat
spherical coefficients of F_model
- crossrot_dns.dat
rotation function map
- molrep_fob.dat
F_observed
also (if you started from MTZ file):
- molrep_mtz.cif
formatted CIF file of F_observed
also (if you used keyword FILE_S):
- align.pdb
corrected by sequence alignment input model
WHAT MOLREP CAN DO¶
+-- Self RF (FUN=R, without any model)
!
+-- Standard MR -+-- Cross RF (FUN=A or FUN=R )
! !
! +-- Locked Cross RF ( FUN=A or R and LOCK=Y )
! !
! +-- TF (FUN=A or FUN=T )
!
!
! +- identical models
! !
! +--Multi-monomer-+
! ! search !
! ! (DYAD=M) +- complex with pairs of
! ! different models
! !
! ! +- two identical models
! ! !
+-- Multi-copy search -+-- Dyad search -+
! for MR ! (DYAD=D) !
! ! +- two different
! ! models
! !
MOLREP --+ +-- Multy-copy for one model
! (DYAD=Y)
!
!
! +-- RF and PTF
! ! (PRF=N)
! !
+-- Fitting two models -+-- SAPTF, RF and PTF
! ! (PRF=S)
! !
! +-- SAPTF, PRF and PTF
! (PRF=Y)
!
!
! +-- RF and PTF
! ! (PRF=N)
! !
+-- Searching in ED map -+-- SAPTF, RF and PTF
! ! (PRF=S)
! !
! +-- SAPTF, PRF and PTF
! (PRF=Y)
!
+-- Rotate and position the model (FUN=S FILE_T)
!
!
!
+-- Search model orientation in electron density map
! for particular position by phased RF (PRF=P FILE_T2)
!
!
! +-- find HA positions by MR solution
! ! (FUN=D, model_2)
! !
+-- HA search ---+-- HA search for SIR or SAD
! ! (DIFF=H, FUN=T, without any model)
! !
! +-- Self RF for HA position
! (DIFF=H, FUN=R, without any model)
!
+-- pure RB refinement (phased or unphased, FUN=B, model_2)
where: FUN, DYAD, PRF, LOCK, DIFF - keyword
MR - Molecular Replacement
RF - Rotation function
TF - Translation function
PRF - Phased Rotation function
PTF - Phased Translation function
SAPTF - Spherically Averaged Phased Translation function
ED - Electron density
HA - heavy atom
RB - rigid body
Search model orientation by PRF
Standard molecular replacement method¶
The program performs molecular replacement in two steps:
- Rotation function (RF)
search orientation of model
The result of the Rotation function depends on the radius of a spherical domain in the centre of the Patterson function (the so-called cut-off radius). This radius must be chosen so as to maximize the ratio between the number of inter- and intramolecular vectors. The program chooses the value of this radius as twice the radius of gyration, but can also use an input value (keyword RAD).
Instead of computing RF, the program can use a list of orientations from a Rotation function (keyword FILE_T) which was prepared before. Anisotropic correction of data before computing RF can be useful for data with high anisotropy (keyword ANISO).
With a second fixed model (MODEL_2), the use of modified stucture factors instead of |Fobs| for RF (keywords DIFF, P2) may make RF clearer. The modified stucture factor is:
sqrt(|Iobs-Imod2*(P2/100)|)
where P2 is the percentage of model_2 in the whole structure.
The Translation function can check several peaks of the rotation function (NP) by computing a correlation coefficient for each peak and sorting the result. For scaling observed and calculated structure factors, the program uses the scaling by the origin peak of Patterson, but for data with high anisotropy the program can use anisotropic scaling (ANISO). The Translation function can take into account the second fixed model (MODEL_2) and also, if the number of monomers is known, MOLREP can position the input number of monomers in a simple run (keyword NMON). Also in this case the possibility to choose from symmetry-related models closest to which was found before is useful (keyword STICK).
The program can detect and use pseudo-translation vectors. In this case the pseudo-translation related copy will be added to the final model (keyword PST).
The Packing function is very important in removing wrong solutions which correspond to overlapping symmetry-related or different models (keyword PACK).
Be careful, with keyword SURF=’Y’ program use to calculate Packing Function only atoms which lying inside of molecule.So, for nonglobular model (for example, only CA atoms) you cannot use keyword SURF=’Y’ with PACK=’Y’.
Use keywords:
Self Rotation Function only
If you define only a file of structure factors (Fobs), the program will compute a Self Rotation function with default value of cut-off radius. Use keyword RAD if you want another value. Other useful keywords: RESMAX, RESMIN, SIM.
Resulting output:
- molrep_srf.tab
List of peaks of Self Rotation Function. molrep_srf.tab is default name, you can use another name using keyword FILE_TSRF.
- molrep_rf.ps
PostScript file of Self Rotation Function which contains four plots RF (theta,phi,chi) for chi = 180, 90, 120, 60. You can change the fourth value of chi (60) by keyword CHI You can change the scale of these plots of RF by keyword SCALE This picture can help understand stereographic projection
Search model in electron density or EM map
In some cases it is difficult to solve an X-ray structure by molecular replacement even when a structure for a homologous molecule is khown. If prior phase information either from SIR/MAD or from a partial structure is known, this could be used in a six-dimensional search. The program divides the six-dimensional search with phases into three steps:
a spherically averaged phased translation function (SAPTF) is used to locate the position of the molecule or its fragment. It compares locally spherically avaraged experimental electron density with that calculated from the model and tabulates highest scoring positions.
then for each such position a local phased rotation function (PRF) is used to find the orientation of the molecule. Another possibility is to use usual rotation function (RF) for modified map, i.e program sets 0 the density outside of sphere with radius = twice radius of model and with the centre in current point.
the phased translation function (PTF) for found orientation which checks and refines the found position.
You need to have the phases in a CIF file of structure factors or to use corresponding keywords for MTZ file or use EM map as input instead of Fobs file. In EM case map will be converted into !F! and phases.
For input map use keywords:
with keyword PRF = ‘N’ (default value): usual Rotation function and Phased Translation function will be used.
with keyword PRF = ‘Y’: SAPTF (Spherically averaged phased translation function), Phased Rotation function and Phased Translation functions will be used.
with keyword PRF = ‘S’: 1.SAPTF (Spherically averaged phased translation function). 2.For current point of SAPTF solution input map is modified, i.e program sets 0 the density outside of sphere with radius = twice radius of model and with the centre in current point. 3.usual Rotation function for this modified map. 4.Phased Translation functions
SFCHECK can convert input map to scaled and/or inverted map.
If you structure contains several molecules which forms some point group you can use this NCS. Program will generate complete model and use it for stage PTF. Also result (molrep.pdb) will be complete model.
First of all you must define parameters of NCS in PDB file or using keywords NCS, ANGLES, CENTRE. See How to define NCS
Other useful keywords:
Also you can refine solution by Pure Rigid Body Refinement
Search model orientation in electron density map for particular position by PRF
You can use this possibility (keywords PRF <#PRF>`__=P and `FUN <#fun>`__=R or A) if you want to find the model orientation in ED map by rotating model around the defined point in ED map. Program puts the origin of model coordinate sysytem to the defined point and performs phased rotation function (PRF). Use keyword `RAD to define the radius of sphere for PRF.
You must define the list of defined points of ED map using file FILE_T2 , wich must contain lines with “Sol_”, peak number,Polar angles and shift (sx,sy,sz) e.g.:
"Sol_ 23 10.0 22.2 40.0 .564 .443 .032"
But program will use only the shift (sx,sy,sz).
Model is rotated around the origin of model coordinate sysytem. If keyword `SURF <#surf>`__= Y,A,2,O program puts the centre of model to the origin of model coordinate sysytem automatically. If you want, for example, to rotate the model around some atom, shift the origin to this atom and use `SURF <#surf>`__=N
Other useful keywords:
Fitting two atomic models (FM)
The idea is to fit the electron densities instead of the atomic models, trying to find the best overlap. Advantages are:
can be used for cases with very low homology;
can be used when amino acid sequence is absent;
no need to use the list of equivalent atoms.
If you define only two files of models (searching model and model_2), without a file of structure factors (Fobs), the program will fit the search model (keyword FILE_M) to the second model (keyword MODEL_2). The search model must be smaller or equal to the second model.
with keyword PRF = ‘N’ (default value): usual Rotation function (RF) to search the orientation and Phased Translation function (PTF) to search position will be used.
with keyword PRF = ‘Y’: Spherically averaged phased translation function (SAPTF) gives the expected position for model. Phased Rotation function (PRF) for expected position gives orientation. Phased Translation function (PTF) checks and refines the translation vector.
with keyword PRF = ‘S’: 1.SAPTF (Spherically averaged phased translation function). 2.For current point of SAPTF solution input map is modified, i.e program sets 0 the density outside of sphere with radius = twice radius of model and with the centre in current point. 3.usual Rotation function for this modified map. 4.Phased Translation functions
Other useful keywords:
The result is file molrep.crd (or molrep.pdb ) - model fitted to second model.
Just rotate and position the model
This possibility may be useful if you want to place the model to a particular orientation and position, or to compare several solutions.
Use keyword FUN=S and define three files: a model (keyword FILE_M), a file of structure factors (keyword FILE_F) and file with polar angles and shifts (keyword FILE_T). The program will shift the model to the origin, rotate (by polar angles) and the position it (in fractional unis). The new model will be written to an output coordinate file. Also the program will compute an R-factor and a Correlation Coefficient.
If you like to use Eiler angles use “Sol_A” instead “Sol_” see molrep_rf.tab
If you like to rotate around some atom, you have to shift coordinate system origin to this atom by hand and use keyword SURF = N.
Other useful keywords:
Multi-copy search
There are three modes: “dyad_search”, “Multi-copy search”and “Multi-monomer search”.
Dyad_search - Search two copies of a model simultaneously (keyword DYAD=D).
Sometimes you can not find a solution starting with one molecule if you have several copies of the molecule in the asymmetrical part of the unit cell. In this case a search with two independent molecules may give a solution. The central point of method is the construction of a multi-copy search model from properly oriented monomers using a special TF (STF), which gives the intermolrecular verctor between properly oriented monomers (dyad). This dyad can then be used for a positional search with a conventional TF.
the program checks all pairs of NP peaks of the Rotation Function (RF). For each pair the program uses the first rotation to prepare model-1. Model-2 will be prepared by using the second rotation and one rotation from the crystallographical symmetry operators. The total number of pairs to be checked is ((NP+1)*NP*Nsym)/2
next, for model-1 and model-2: the program computes the Special Translation Function ( STF) to find the inter-molecular vector of the dyad.
for NPT peaks ( i.e. inter-molecular vectors) of the STF, the program computes the standard Translation Function (TF) using the current dyad as a model, and it calculates a Correlation Coefficient for the first`NPTD <#dyad_nptd>`__ peaks of the TF.
Solution and output file: molrep.pdb will be the dyad with the best Correlation Coefficient (or several dyads if keyword NMON > 1).
WARNING: the procedure takes quite some time, because the total number of Translation Functions to be calculated is NMON*NPT*((NP+1)*NP*Nsym)/2.
In the output .log (.doc) file you can find the following information:
Sol_ R1 R2 Rs Rslf STF TF Shift_1 PFmax PFmin Rfac Corr Sol_ 1 1 1 0 2 1 0.059 0.000 0.201 1.01 0.99 0.569 0.379 and Sol_best 1 1 1 0 2 1 0.059 0.000 0.201 1.01 0.99 0.569 0.379 Sol_best Rot1-->2 Dyad_vector dist d_ort d_par Sol_best 0.0 0.0 0.0 -0.210 0.000 -0.487 39.2 19.6 33.9
These lines means:
- R1
peak number of rotation for model-1
- R2
peak number of rotation for model-2
- Rs
CS operator number which applyed before rotation for model-2
- Rslf
peak number of self rotation function
- STF
peak number of special translation function
- TF
peak number of translation function
- Shift_1
position of model-1
- PFmax PFmin
min, max values of Packing function
- Rfac Corr
R-factor and Correlation Coefficient
- Rot1->2
polar angles of rotation from model-1 to model-2.
- Dyad_vector
vector (in fractional) from model-1 to model-2.
- dist d_ort d_part
first number - distance between models (in Angstrom) second number - distance orthogonal to rotation 1->2 third number - distance parallel to rotation, i.e. for pure dimer this is 0.
Also you can find additional information:
Sol_ angles_1 angles_2 shift_2 Sol_ 90.63 98.70 118.12 90.63 98.70 118.12 0.189 0.256 -0.415 +---------------------------------------------------------+ ! ! ! ! ! ! ! ! ! ! ! ----------------- ----------------- ! ! / / ! ! / / ! ! ! rotated (angles_1) ! ! rotated (angles_2) ! ! ! ! monomer_1 ! ! monomer_2 ! ! ! ! ! dyad ! ! ! ! ! +----------!-----------------+ ! ! ! ! / ! vector! ' ! ! ! ! / / ' / ! ! / / / ! ! / / ' / ! ! ---/------------ ' -------------- ! ! /shift_1 ' shift_2 ! ! / ' ! ! / ' ! ! / ' ! ! / ' ! ! / ' ! ! / ' ! ! / ' ! !/ ' ! +---------------------------------------------------------+ origin
If you believe the Self-RF, you can try to find a dyad which has the rotation between monomers corresponding to the rotation of the Self-RF (use keywords NSRF, FILE_TSRF).
Model-2 can be different from model-1. Use keywords FILE_M2 to define file of searching model-2, FILE_T2 with list of peaks rotation function for this model (this RF have to be computed before) and NP2 number of peacks which will be used.
Multi-copy search - Search many copies of a model (not only dyad) (keyword DYAD=Y).
Program starts to search a single monomer, after that produces the dyad search, repeates dyad search for next dyad with the first being fixed and ,finaly, tryes add a single monomer.
Multi-monomer search - construct complexes of monomers and check. (keyword DYAD=M).
Instead to search two copies and then next one program constructs maximal possible complexes of monomers (3mres,4mers,5mers,…) and checks its by TF. This algorithm is better and faster then DYAD=D or DYAD=Y. But also it takes a time (about 30min,1h,2h …). It depends on resolution, size of cell, space group.
Use keywords:
For DYAD M:
and also:
Model correction
You can improve your model beforehand by using keyword SURF.
N
do not perform any model correction.For FUN=S (just_rotate_and_position) program changes N to O
O
only shift to the origin
A
make the protein into a polyalanine model ( i.e. remove from the model: water molecules, H atoms, atoms with alternative conformation (except the first), atoms with occupacy = 0), make all B = 20, and shift to the origin
Y
remove various atoms from the model (water molecules, H atoms, atoms with alternative conformation (except the first), atoms atoms with occupacy = 0), shift to the origin, compute atomic accessible surface area and replace atomic B with B = 15.0 + SURFACE_AREA*10.0
2
set all B = 20 and shift to the origin
C
as Y but new B only for Packing function (not change original B) and shift to the origin
Using sequence alignment
Another way to improve your model is to use the sequence of the unknown structure.
Use keyword FILE_S to define a file containing a sequence. This sequence file must be ASCII:
!> title !# sequence !SVIGSDDRTRVTNTTAYPYRAIVHISSSIGSCTGWMIGPKTVATAGHCIY !# this is comment ! DTSSG--SFAGTATVSP GRNGTSYPYG !NRGTRITKEVFDNLTNWKNSAQ !
If the first symbol in the line is “#”, it means the line contains comments. Blancs are ignored.
The program will perform sequence alignment and create a new corrected model with the atoms corresponding to the alignment. The output file with the corrected model is align.pdb. Without an Fobs file, the program only performs model correction.
NMR Model
You can use PDB file with NMR models or pseudo-NMR file with several homologous structures which were superimposed before. Algorithm is equivalent to sum RF or/and TF for individual structures. Program can find the best model in NMR file or use all models (see keyword NMR) .
In the PDB file different models must be separated by MODEL record. For example:
HEADER HYDROLASE (ENDORIBONUCLEASE) CRYST1 64.900 78.320 38.790 90.00 90.00 ... MODEL 1 ATOM 1 N ASP A 1 45.161 12.836 ... ATOM 2 CA ASP A 1 45.220 12.435 ... ... ATOM 745 SG CYS A 96 58.398 6.673 ... ATOM 746 OXT CYS A 96 62.238 7.178 ... ENDMDL MODEL 2 ATOM 1 N ASP B 1 44.487 11.386 ... ATOM 2 CA ASP B 1 44.559 11.129 ... ...
Use keyword NMR
EM or electron density model
Searching model can be Electron Microscopic model (EM) or electron density map. Only values higher the limit (if keyword ROLIM is defined) will be used. Map must have space group P1 and contains whole model. Vector ORIGIN defines the centre of model and the rotation will be performed around this point. If parameter DRAD (radius of model) is defined program will use the density only inside the sphere with radius = DRAD and with centre in vector ORIGIN.
+--------------------------------+ nz
! ! !
! . .
!
! ! !
! . .
!
! ! !
! +--------------------------------+ izmax
! ! !
! ! !
! ! !
! ! ---------------- !
! ! / !
! ! / !
! ! / !
C_cell ! / !
! ! ! ! !
! ! ! DRAD ! !
! ! !---------- + ! !
! ! ! / centre ! !
! ! ! / / !
! ! / / !
! ! / / !
! ! / / !
! ! / / !
! ! -/-------------- !
! ! / !
! ! / !
! ! / ORIGIN !
! ! / !
! ! / !
! ! / !
! !/ !
! +--------------------------------+
0 nx
----------- A_cell --------------
Program will get vector ORIGIN from file automatically. If it is not possible to get correct vector, program will use ORIGIN = ( 0.5, 0.5, izmax/(2*nz)). If you want you can define ORIGIN yourself.
Use keywords:
Search model in electron density map will be performed as usual.
Locked Cross Rrotation Function
Locked Cross Rotation function (LRF) means to average the Cross Rotation function by NCS which can be determined with Self Rotation function. LRF is especially useful when NCS forms a group.
Use keywords:
Rigid body refinement
You can use
Rigid Body Refinement in
Patterson or Real space (keyword FUN = ‘B’). This possibility is useful in the last stage of MR. For example after fitting the model into EM map. You must define Fobs or Fobs and phases (or Map). Also use keyword MODEL_2 for model to refine. If you define the phases or use the map program will produce real space refinement (more correctly, in reciprocal space using phase information).
If keyword DOM = ‘N’ (default) program refines MODEL_2 as single molecule.
If keyword DOM = ‘Y’ program performs multi-domain refinement. For this you must put into PDB file additional lines before each domain. Additional line contains word ‘#DOMAIN’ and domain number (free format).
For example:
HEADER HYDROLASE (ENDORIBONUCLEASE) CRYST1 64.900 78.320 38.790 90.00 90.00 ... #DOMAIN 1 ATOM 1 N ASP A 1 45.161 12.836 ... ATOM 2 CA ASP A 1 45.220 12.435 ... ... ATOM 745 SG CYS A 96 58.398 6.673 ... ATOM 746 O CYS A 96 62.238 7.178 ... #DOMAIN 2 ATOM 747 N PHE A 97 44.487 11.386 ... ATOM 748 CA PHE A 97 44.559 11.129 ... ... ATOM 945 C VAL A 196 58.398 6.673 ... ATOM 946 O VAL A 196 62.238 7.178 ... #DOMAIN 1 ATOM 947 N ASP A 197 44.487 11.386 ... ATOM 948 CA ASP A 197 44.559 11.129 ... ...
If you structure contains several molecules which forms some point group you can use this NCS as constraints. First of all you must define NCS parameters in PDB file or using keywords: NCS,ANGLES, CENTRE. See How to define NCS
If you have some trouble with NCS parameters (values: theta,phi,chi,cx,cy,cz) use keyword DOM = ‘I’ and you can find these actual values in output PDB file: molrep.pdb. Alternative way to create initial PDB file with complete model is to describe only first (reference) molecule and use keyword DOM = ‘C’. Complete model you can find these in output PDB file:molrep.pdb. Finally use keyword DOM = ‘S’ for refinement with constraints.
Use keywords:
Find HA positions by MR solution
To define derivative use corresponding label for MTZ file or derivative file (FILE_DER).
Use keywords:
Heavy atom search
To define derivative use corresponding label for MTZ file or derivative file (FILE_DER).
In this case you need not to use any model.
Use keywords:
HOW TO USE MOLREP¶
(you can use Testing_program_MOLREP as tutorial)
A simple way to use MOLREP is to define files for Fobs and the model and use default values for all parameters ( i.e. without using any keywords). There is always a chance of solving the structure automatically. If this does not work, use a common strategy of molecular replacement.
Planning ahead¶
Success of the molecular replacement method depends on:
quality of experimental data
low resolution limit
high resolution limit
quality of the model, homology, conformation
Things to look out for:
- data
Look at your data quality. Completeness is very important. Absence of low resolution reflections may cause problems, especially if the model is some part of a whole structure. Look at anisotropy and twinning. Think carefully: can you ‘safely’ use the high resolution reflections? If not, use keyword SIM to remove the potentially bad effect of this part of the data. It might be a good idea to use some program to check the data, for example SFCHECK.
- model
Look at the model regarding the shape. The automatic choice of the cut-off radius for RF is twice the radius of gyration. This is good enough if the shape is approximately spherical. If the model is very asymmetrical, it is better to make a choice yourself. Make a choice for SIM, RESMIN. If you have not any idea about similarity, SIM=0.35 is a good approximation. If you have a dimer use it, but use RAD corresponding to a monomer. It is very useful to shift the model to the origin of coordinates. Use keyword SURF = O or Y (Y is default).
- Self-RF
Compute Self-RF. It may give you some idea about NCS or about the number of copies in the asymmetrical part of unit cell.
- Cross-RF only
In the DOC_file you can find the list of expected orientations of the model and also the rotations between them. Compare this with the Self-RF. This is an additional check of correctness of the expected orientation. But sometimes we can not find corresponding peaks in Self-RF for correct orientation.
- Translation function
If there are several copies of the model in the asymmetrical part of unit cell, use keyword NMON or multi-copy or dyad search. It is not necessary to use the option of Pseudo-translation for a dyad search, since this can recognize Pseudo-translation itself.
Dialogue or batch¶
You can use MOLREP by dialogue or by command (batch) file. Modern computing technology allows the carrying out of most of the calculations for small and medium sized proteins in real time, therefore, dialogue is a preferable way of running MOLREP. However, the program automatically produces a batch command file during dialogue. This feature might be useful for repeated calculations.
Pseudo-translation¶
MOLREP can detect pseudo-translation, and define a pseudo-translation vector.
If keyword PST = Y, the program applies pseudo-translation with a pseudo-translation vector which was defined by the program or the user. When calculating a Translation Function, the program will use this vector to modify structure factors. Pseudo-translation copy will be added to the final model at the end program running.
If FUN=R and LIST=L MOLREP computes a list of Patterson peaks and writes these to molrep.doc. This may be helpful in the detection of pseudo-translation.
Use keywords:
Flexible model¶
If your model is flexible, for example, consists of two domains, you can try to solve this problem by two ways:
1. Create two files for each domain and use dyad search (DYAD = D or DYAD = M)
2. Solve one domain, refine it and use the solution as fixed model to solve another domain.
The use many homologous models¶
If you have several homologous models you can create a pseudo NMR file with these models and use its together (see NMR model). But these models must be superimposed before, for example, by MOLREP (see fitting two models).
Keep in mind¶
Without a model file, the program only computes a Self Rotation Function.
If FUN=R, the program computes and writes to molrep.doc all symmetry-related peaks of the Rotation Function. If also keywords NSRF and FILT_TSRF are used you can fine the pairs of peaks of cross RF which corresponds to NCsymmetry.
If you want to change the space group of the structure factor file, use keyword NOSG, i.e. new space group number, or keyword SG
The Packing Function (PF) is very important to remove wrong solutions which correspond to overlapping symmetry-related or different models. But you can remove this option (PACK = N ), for example, if you want to find the model in a special position. Value of PF = 1 corresponds to non-overlapping, value = -1 corresponds to completely overlapping two models.
When MOLREP is trying to find several models (NMON > 1) it is useful to use keyword STICK = Y. Then for each additional molecule the program will choose a symmetry-related molecule closest to which was/were found before.
For the PRF and the SAPTF, the default cut-off radius is once the radius of gyration, whereas for a Patterson calculation the cut-off radius would be twice the radius of gyration.
Tips:¶
- 1
In the beginning use molrep without keywords or only SIM (default 0.35)
- 2
In case with pseudo-translation use also “PST N”
- 3
If input model is an ensemble of models (for example from BALBES) try also “NMR 1” (some variant of RF) and “NMR -1” means only first model from ensemble.
- 4
If you are searching additional part of structure in the map after refmac: “molrep -f refmac.mtz -m model.pdb -mx refmac.pdb” with “LABIN F=FWT PH=PHWT” use “DIFF M”,”SIM -1” (i.e. not use SIM), NP about 50 also you can try “PRF Y” or “PRF S” (SAPTF)
- 5
In difficult case play with SIM, RESMIN and NP
- 6
To check alternative space groups use “SG ALL”
- 7
For “DYAD M” useful keywords are “ALL”,”NPTD” and “NML”
- 8
You can stop program safely if you create in current directory or in PATH_OUT (if option “-po” is used) file: molrep_stop_signal.xml (contents does not matter)
KEYWORDED INPUT¶
It is easy to use MOLREP interactively, but can be used in batch. The available keywords are:
Common:
FILE_T, FUN, NMON, NP, NPT, RAD PATH_SCR
And for structure factors control:
And for model control:
And for multi-copy search:
DYAD, FILE_M2, FILE_T2, NP2, NPTD, NSRF
And for search in ED:
And for fitting two models:
And for EM or electron density model:
DSCALEM, INVERM, ROLIM, DRAD, ORIGIN
And for EM or electron density instead of Fobs:
Common:
ANISO, BADD, LIST, LMAX, LMIN, PACK,
And for standard MR:
DIFF, FILE_S, NMR, NOSG, P2, PST, STICK, VPST, LOCK,
And for Self RF:
And for multi-copy search:
AXIS, DIFF, DIST, P2, ALL, STICK
And for search in ED:
And for fitting two models:
And for search in ED:
And for Pure Rigid Body Refinement:
General keywords¶
NP <np>¶
Default: <auto>
<np> is the number of peaks from the rotation function to be used (maximum: 200).
NPT <npt>¶
Default: <15>
<npt> is the number of peaks from the translation function to be used (maximum: 50).
NMON <nmon>¶
Default: <auto>
<nmon> is the number of monomers. The program will try to create a full model, which will consist of NMON initial models plus model_2.
RESMIN <resmin>¶
Default: <auto>
soft resolution cut_off: Boff =2*RES_min^2
COMPL <compl>¶
Default: automatic choice
COMPL = V_model/(V_cell/Nsym), RESMIN ~ Rad_model. It corresponds to Boff:
SIM <sim>¶
Default: 0.35
Similarity of the model: from 0.1 to 1.0. It corresponds to Badd. SIM=1 means normalized F will be used. SIM=-1 means do not use SIM (i.e. BADD=0) If SIM is used, the keyword BADD is ignored.
- SIM (Badd)
controls high resolution data
- RESMIN,COMPL (Boff)
controls low resolution data
The use of Boff and Badd means to change Fobs and Fmodel:
FUN < A | R | T | S | B | D >¶
Default: <A>
R |
calculate only Rotation Function |
T |
calculate only Translation Function, reading list of peaks of RF from file (molrep_rf.tab) or from TAB_file |
A |
calculate both: RF and TF |
S |
rotate and position the model and compute R-factor and Correlation Coefficient |
B |
pure Rigid Body refinement |
D |
find HA positions by MR solution |
FILE_T <filename>¶
Default: <molrep_rf.tab>
Input or output TAB_file (see also molrep_rf.tab)
MODEL_2 <filename>¶
Default: no model_2
Input file with the second (fixed) model in correct position and orientation, in PDB or BLANC format. This model will be fixed during the search. When fitting two models to each other, the second model is the target model.
SURF < N | Y | A | O | 2 | C >¶
Default: <Y>
Perform model correction.
N
do not perform any model correction.For FUN=S (just_rotate_and_position) program changes N to O
O
only shift to the origin
A
make the protein into a polyalanine model ( i.e. remove from the model: water molecules, H atoms, atoms with alternative conformation (except the first), atoms with occupacy = 0), make all B = 20, and shift to the origin
Y
remove various atoms from the model (water molecules, H atoms, atoms with alternative conformation (except the first), atoms atoms with occupacy = 0), shift to the origin, compute atomic accessible surface area and replace atomic B with B = 15.0 + SURFACE_AREA*10.0
2
set all B = 20 and shift to the origin
C
as Y but new B only for Packing function (not change original B) and shift to the origin
RAD <rad>¶
Default: automatically calculated from the model, unless:
in case of Self-RF calculations: 30Å
for Rotation Function calculations: twice the radius of gyration
for PRF and SAPTF: radius of gyration
Cut-off radius for Patterson search or for electron density search.
RESMAX <resmax>¶
Default: <auto>
High resolution limit.
PATH_SCR <path>¶
Default: <not used>
Path to directory for scratch files. For example: /y/people/alexei/
Keywords for special cases
PST < N | C | Y >¶
Default: <N>
How to deal with pseudo-translation.
N |
ignore pseudo-translation altogether |
C |
check only, but do not use pseudo-translation |
Y |
use pseudo-translation. |
VPST <vpst1,vpst2,vpst3>¶
Default: automatically from Patterson
Pseudo-translation vector (in fractional units), used when PST = Y.
BADD <badd>¶
Default: <0>
ANISO < N | Y | C | S | K >¶
Default: <N>
N |
do not use anisotropic correction and/or scaling |
Y |
|
C |
use anisotropic correction of Fobs for RF only |
S |
use anisotropic scaling for TF only |
K |
use scaling without B-factor |
PACK < Y | N >¶
Default: <Y>
Y |
use Packing Function with Translation Function |
N |
do not use Packing Function with Translation Function |
LMIN <lmin>¶
Default: <4>
Minimum L-index of spherical coefficients. The program does not use coefficients with L=0. Possible values are 2,4,6,… L = 2 means to use all coefficients up to Lmax.
LMAX <lmax>¶
Default: automatic choice
Maximum L-index of spherical coefficients. Possible values are 2,4,6,8,…,98,100.
PRF < N | Y | S | P >¶
Default: <N>
N |
standard RF and Phased Translation Function is calculated |
Y |
SAPTF (Spherically averaged phased translation function), Phased Rotation Function (PRF) and Phased Translation Function will be used. |
S |
SAPTF (Spherically averaged phased translation function), Usual Rotation Function (RF) for modified map and Phased Translation Function will be used. |
P |
Search the model orientation in ED map by rotating model around the defined points in ED map. List of points must be in the file FILE_T2. |
Program will use the phases of BLANC (by keyword PHASE) or from MTZ file or from EM map.
If keyword FUN=T, rather than computing the Rotation Function, the program reads rotation function results from file FILE_T ( or “molrep_rf.tab”): “Sol_ peak number, polar angles (theta,phi,chi) and shift (sx,sy,sz)”
PHASE < name >¶
Default: none
BLANC file of phases. If input Fobs file is CIF use ‘PHASE +’. It means to use the phases from CIFile.
NOSG <nosg>¶
Default: <0>
Number of new space group if you want to change the space group for the file of structure factors. Program just changes space group name, group number and cryst. symmetry operators, but not cell and data.
SG <name>¶
Default: < >
New space group name. “ALL” means to check all possible SG.
DIFF < N | P | F | M | H >¶
Default: <N>
N |
use unmodified structure factors |
P |
use modified stucture factors instead of Fobs for RF, as follows: sqrt(|Iobs-Imod2*(P2/100)|) |
F |
use modified stucture factors instead of Fobs for RF, as follows: vector difference (Fobs - Fmod2*(P2/100)) |
M |
remove fixed model from diff. fourie by mask (model_2). |
H |
for heavy atom search |
P2 <p2>¶
Default: <0>
Percentage of model_2 in the structure.
NREF <ncycle>¶
Default: <5>
number of cycles of rigid body refinement.
STICK < N | Y >¶
Default: <Y>
Choose from symmetry-related models closest to which found before (this option does not work with pseudo-translation possibility).
FILE_S <filename>¶
File with sequence for model correction by sequence alignment.
NMR < 0 | 1 | 2 | 3 >¶
Default: <0>
0 |
use PDB file with NMR structures as single model |
1 |
use NMR possibility only for RF |
2 |
use NMR possibility for RF and TF. Best NMR model will be found and used as solution. |
3 |
use NMR possibility for RF and TF. Averaged TF will be used. All NMR models will be used as solution. |
LOCK < Y | N >¶
Default: <N>
Locked Cross Rotation function will be performed. Use also keywords: FILE_TSRF and NSRF
Without FILE_TSRF program computes Self_RF and uses NSRF peaks from it.
SELF < N | A >¶
Default: <N>
A means after RF to compute Self_RF and use from RF only related peaks for TF.
If you like you can use keyword: NSRF
SCORE < Y | N | C >¶
Default: <Y>
N |
do not stop if contrast is good |
C |
Corr.Coef. instead Score and do not stop |
Keywords specific for multi-copy search
DYAD < N | Y | D | M >¶
Default: <N>
Y |
multi-copy search |
D |
dyad search |
M |
multi-monomer search |
DIST <Dmin,Dmax,Dpar>¶
Three distances for dyad search.
Dmin |
Default: radius of gyration. minimal distance between molecules |
Dmax |
Default: 1000Å. maximal distance between molecules |
Dpar |
Default: 1000Å. maximal shift along rotation axis |
AXIS <Chi,Delta>¶
Default: <0,0>
- Chi
check only rotation by Chi (in degrees). 0 means to check all orientations.
- Delta
delta for Chi (in degrees)
NSRF <nsrf>¶
Default: <0>
Number of peaks of Self-RF which will be used. 0 means not to use Self-RF. A list of Self-RF peaks will be taken from file defined by keyword FILE_TSRF which must be prepared in advance (see Self Rotation Function).
NPTD <nptd>¶
Number of peaks in the STF (Special Translation Function) to be used.
NPT <nptd>¶
Number of peaks in TF to be used.
NP2 <np2>¶
Number of peaks in RF for second searching model to be used for dyad search.
FILE_M2 <filename>¶
file of second searching model
FILE_T2 <filename>¶
file with list of peaks of RF for second searching model
ALL < N | Y >¶
Default: <N>
if ALL = Y , program will use all Crystallographical Symmetry Operators
NML <nml>¶
number of Nmers to check (DYAD = “M”).
Keywords for Self Rotation Function
Without a file of the model, the program computes a Self Rotation Function.
CHI <chi>¶
Default: <60>
Angle chi of additional fourth section of RF(theta,phi,chi).
SCALE <scale>¶
Default: <6>
Maximum value of RF is SCALE * SIGMA(RF).
FILE_TSR <filename>¶
Default: <molrep_srf.tab>
Input or output TAB_file with peaks of Self_RF.
Keywords for EM or electron density model:¶
DSCALEM <scale>
Default: <1>
scale factor of correction of density cell
INVERM < N | Y >¶
Default: <N>
If Y , inverted phases will be used
ROLIM <limit>¶
Default: <not used>
minimal value of density which will be used
DRAD <radius>¶
Default: <0>
radius of the model (in A). If parameter DRAD is defined program will use the density only inside the sphere with radius = DRAD and with centre in vector ORIGIN.
ORIGIN <vector>¶
Default: <0,0,0>
center of the model in the cell (in fract.units)
Keywords for EM or electron density instead of Fobs:¶
DSCALE <scale>¶
Default: <1>
scale factor of correction of density cell
INVER < N | Y >¶
Default: <N>
If Y , inverted phases will be used
DLIM <limit>¶
Default: <not used>
minimal value of density which will be used
Keywords for Rigid Body Refinement:¶
DOM < N | Y | I | S | C >¶
Default: <N>
N
RB refinement as single body.
Y
Multi-domain refinement.
I
Give only information about molecule-domain structure. Useful for RB refinement with constraints.
S
Multi-domain refinement with constraints.
C
only create complete model using NCS parameters. See How to define NCS
Keywords for NCS parameters:¶
NCS <ncs_id>¶
Default: <0>
NCS identifier or = “1” which means to use NCS parameters from file.
ANGLES <theta,phi,chi>¶
Default: <0,0,0>
Polar angles of NCS which define the standard system orientation in the cell.
CENTRE <vector>¶
Default: <0,0,0>
position of the NCS centre in the cell (in fract.units)
Keywords for Finding or Searching Heavy atoms:¶
FILE_DER <filename>¶
Input file of derivative or use labels for MTZ file.
COMMAND (BATCH) FILE¶
The best and easiest way to prepare a command file is to run MOLREP once by dialogue. The program creates a command (batch) file ( molrep.btc ) automatically.
See some command (batch) file examples.
MOLREP VERSION TO READ MTZ file¶
Keywords for reading MTZ file¶
The following keywords are necessary only for MTZ files.
F |
label of F or F(+) |
SIGF |
label of sigma F or sigma F(+) |
F(-) |
label of F(-) |
SIGF(-) |
label of sigma F(-) |
I |
Label of Intensity of hkl |
SIGI |
Standard deviation of the above |
I(-) |
label of Intensity of -h -k -l |
SIGI(-) |
Standard deviation of the above |
PH |
Label of phases |
FOM |
Label of figure of merit |
FD |
Label of F-derivative |
SIGFD |
Label of sigma F-derivative |
DP |
Label of !F(+)!-!F(-)! |
SIGDP |
Label of DP |
TESTING THE MOLREP PACKAGE¶
(also you can use this as tutorial)
In directory “../molrep/molrep_check/” there are two files:
…/molrep/molrep_check/mr.bat - command (batch) X-ray testing
…/molrep/molrep_check/em.bat - command (batch) EM testing
In directory “../molrep/molrep_check/data/” there are several files for X-ray test:
In directory “../molrep/molrep_check/em/” there are several files for EM test:
See “../molrep/molrep_check/readme”
MOLECULAR REPLACEMENT METHOD - THEORY¶
Molecular replacement method (MR)
There are two major steps in the Molecular replacement method: orientation and translation search. They are performed by Rotation and Translation function. Both of them are correlation functions (or overlapping functions) between observed and calculated from model Patterson.
Rotation function ( RF ):
ROT(R) = I Pobs(r) * Pcalc(R,r) dr
rad
where
- R
operator of rotation
I rad
integral inside a sphere in the centre of patterson with radius=rad ( i.e. the cut-off radius)
- P obs
observed Patterson
- P calc
calculated Patterson for rotated ( R ) model
Translation function ( TF ):
TR(s) = I Pobs(r) * Pcalc(s,r) dr =
cell
= Sum ( I Pobs(r) * Pcalc_ij(s,r) dr) = Sum TRij(s)
i#j i#j
where
- s
vector of translation
- I
integral
- i,j
cryst. symmetry operator numbers
- P :sup:`calc_ij`(s,r)
calculated Patterson for model corresponding to ith operator and model corresponding to jth operator
- TR :sup:`ij`(s)
translation function of Pattersons P :sup:`obs`(r) and P :sup:`calc_ij`(s,r).
The Translation Function is the sum of translation functions for each pair of different cryst. symmetry operators.
The best rotation function algorithm is the Crowther Fast Rotation Function which we use here. It utilizes FFT. MOLREP can compute the Rotation Function for three different orientations of the model and average them. That reduces the noise of Rotation function.
Translation function algorithm was developed by the author and performs calculations in the reciprocal space using FFT.
There are two major differences from other translation functions.
Instead of summation of the translation functions for two operators TR ij, we use their multiplication which makes the resulting map far more contrast-rich.
Finally we can multiply the translation function with the Packing Function to remove peaks corresponding to incorrect solutions with bad packing.
Packing function ( PF ):
The overlap for the model in the position s
O(s) = Sum (I Ro_i(r,s) * Ro_j(r,s) dr), where i#j i,j
where Ro_i(r,s) is the electron density of the model which corresponds to the ith cryst. symmetry operator.
Packing function:
P(s) = 1 - scale * O(s)
scale is chosen to make overlap of one copy with itself equal one, i.e. to make
I Ro_i(r,s) * Ro_i(r,s) dr = 1
Finally we have P(s) = 1 for no overlap. Negative values are converted to 0.
If we have a fixed model
O(s) = Sum (I Ro_i(r,s) * Ro_j(r,s) dr) + Sum (I Ro_fixed(r) * Ro_i(r,s) dr)
The algorithm of calculation of the Packing Function is similar to the one for the Translation Function and performed by the same program.
Finally the ‘advanced’ Translation function is:
TR(s) = [ M TRij(s) ] * P(s) i#j
where M means multiplication of different TR ij.
Scaling by Patterson
For scaling we use a completely new strategy based on the Patterson origin peak which is approximated by a Gaussian. This peak is computed for both the observed and calculated amplitudes, and each case the B_overall is computed. The difference
B_diff_overall = B_obs_overall - B_calc_overall
is then added to calculated B_overall so as to make the width of the calculated Patterson origin peak equal to the observed peak. This method makes it possible to have a good approximation for the scaling problem even if only low resolution data is available where other methods do not work. Scaling by Patterson is also useful for the Cross Rotation Function where we have different cells for the model and the unknown structure.
Low resolution cut-off (Boff)¶
Low resolution cut-off introduces systematical errors in the electron density especially near the surface of the model. This is known as the series termination effect. Instead of using the usual low resolution cut-off, MOLREP multiplies the modules of the structure factors by a special coefficient:
Fnew = Fold (1-exp(-Boff*s2)), where Boff= 2resmin2
Boff is called the “soft low resolution cut-off”, which allows removal of structure factors in this resolution range without inroducing the series termination effect.
The use of a priori knowledge of similarity and completeness of the model¶
For low similarity the high resolution reflections are weighted down. For this, MOLREP uses an additional overall factor Badd:
Fnew = Fold exp(-Badd*s2)
Value of similarity ‘SIM’ can be: from 0.1 to 1.0. It corresponds to Badd: from (B_limit-Boverall) to -Boverall, where B_limit + 80.
SIM=1 means normalized F will be used.
For low completeness, e.g. when there are several molecules in the a.u., the contribution of low resolution reflections is weighted down. To manage the completeness of the model, MOLREP uses a low resolution cut-off (Boff). Completeness of model ‘COMPL’ can be : from 0.2 to 1.0. It corresponds to Boff: from 400 to 1600.
Functions of electron density searching ( SM )¶
We suggest a new approach to divide a phased six-dimensional search into three steps:
A spherically averaged translation function is used to locate the position of a molcule or its fragment. It compares locally spherically averaged experimental electron density with that calculated from the model and tabulates highly probable positions accordingly.
Then for each position a local phased rotation function is used to find the orientation of the molecule.
The third step is the phased translation function, used to check and refine the found position.
Spherically averaged phased translation function SAPTF¶
SAPTF gives the expected position of a model in an electron density map by the comparison of spherically averaged density of the model with locally spherically averaged observed density.
SAPTF(s) = I Robs(r,s) * Rcalc(r) dr rad(s)
where
I rad(s)
integral inside a sphere centred in point s of electron density with radius=rad ( i.e. the cut-off radius)
- R obs
spherically averaged around point s observed electron density
- R calc
spherically averaged around origin of coordinate system calculated electron density for model
Phased Rotation function ( PRF )¶
PRF gives the orientation of model placed in some point of electron density.
PROT(O) = I Robs(r) * Rcalc(O,r) dr rad(s)
where
- O
operator of rotation
I rad(s)
integral inside a sphere centred in point s of electron density with radius=rad
- R obs
observed electron density
- R calc
calculated electron density for rotated (O) model
Phased Translation function PTF¶
Translation search in electron density map.
PTR(s) = I Robs(r) * Rcalc(s,r) dr cell
where
- s
vector of translation
- I
integral
- R obs
observed electron density
- R :sup:`calc`(s,r)
calculated electron density for model placed in the vector s
Fitting two models ( FM )¶
Fitting through electron density. Second model (MODEL_2) is the target model which converted to electron density. To search the best overlapping of electron densities of models there are two algorithms:
Rotation Function (Patterson) and Phased Translation Function (electron density).
All functions for electron density. Spherically Averaged Phased Translation Function gives expected position for model. Phased Rotation Function for expected position gives orientation. Phased Translation Function checks and refines the translation vector.
Special Translation Function (
STF ) for dyad search
Multi-copy search¶
Search two copies of a model simultaneously. There are three stages to this:
Rotation function. The program checks all pairs of first NP peaks of Rotation Function (RF). For each pair the program uses the first rotation to prepare model-1. Model-2 will be prepared by using the second rotation and one rotation from the crystallographical symmetry operatators.
Next, for the current pair (model-1 and model-2): MOLREP computes the Special Translation Function (STF) to find the inter-molecular vector of this dyad.
For NPT peaks of the previous Special Translation Function (STF) ( i.e. for NPT inter-molecular vectors) the program computes a standard Translation Function (TF) using the current dyad as model and calculates a Correlation Coefficient for first NPTD peaks of TF.
Special Translation Function (STF)¶
Imagine two models in the asymm. part of the unit cell:
- F1(h)
structure factor of model_1 with the centre of gravity in the origin of the coord. system
- F2(h)
structure factor of model_2
Let
- S1
vector in unit cell from the origin of the coord. system to the centre of gravity of model_1
- S2
vector for model_2
When F(h) is the total structure factor (for the whole crystal structure):
F(h) = F1(h)exp(-2pihS1) + F2(h)exp(-2pihS2)
Then the Patterson is:
P(h) = F(h)*F'(h) = F1(h)*F1'(h) + F1'(h)*F2(h)*exp(-2pih(S2-S1)) + F2'(h)*F2(h) + F1(h)*F2'(h)*exp(-2pih(S1-S2)) = P0(0) + P1(S2-S1) + P1(S1-S2)
The Special Translation Function is a Phased TF with a Patterson function as electron density and P1 = F1’(h)*F2(h) as structure factors of the model. Solution of this function is the dyad vector S1-S2.
Anisotropic correction and scaling
Aniso correction: For Structure Factors we can estimate: 1. isotropic B_overal: F(s) ~ Scale_overall * exp (-B_overall*s^2) 2. anisotropic B_overall (tensor) : F(s) ~ Scale_overall * exp(-(B11a*a*hh +2B12a*b*hk+..) Aniso correction means to make data isotropic with B_overall: F_new(s) = F_old(s) * exp(+(B11a*a*hh +2B12a*b*hk+..) * exp(-B_overall*s^2) Aniso scaling: Fnew = Scale*Fold*exp(-(B11a*a*hh +2B12a*b*hk+..) Scale ans aniso B are taken by mimimization: sum(!Fobs-Fnew!)
INPUT FILE EXAMPLES¶
A. Example of CIF file of amplitudes:¶
data_structure_9ins
_cell.length_a 100.000
_cell_length_b 100.000
_cell.length_c 100.000
_cell.angle_alpha 90.000
_cell.angle_beta 90.000
_cell.angle_gamma 90.000
_symmetry.space_group_name_H-M 'P 1 21 1'
loop_
_refln.index_h
_refln.index_k
_refln.index_l
_refln.F_meas_au
_refln.F_meas_sigma_au
2 3 4 12.3 1.2
-2 -3 -4 11.4 1.1
. . . . . . . . . . . . .
For intensities use:
_refln.intensity_meas
_refln.intensity_sigma
B. Example of CIF file of amplitudes with phases:¶
data_9ins
_cell.length_a 100.000
_cell_length_b 100.000
_cell.length_c 100.000
_cell.angle_alpha 90.000
_cell.angle_beta 90.000
_cell.angle_gamma 90.000
_symmetry.space_group_name_H-M 'P 1 21 1'
loop_
_refln.index_h
_refln.index_k
_refln.index_l
_refln.F_meas_au
_refln.F_meas_au_sigma
_refln.phase_calc
_refln.fom
1 0 0 3468.4934 138.7397 0.746 1.000
2 0 0 618.4012 24.7360 11.948 1.000
. . . . . . . . . . . . . . . . . . . . . . . . . . .
Phases are in degrees.
C. Example of PDB file of amplitudes:¶
HEADER R2SARSF 15-JAN-91
COMPND RIBONUCLEASE SA (E.C.3.1.4.8) COMPLEX WITH 3'-*GUANYLIC ACID
SOURCE (STREPTOMYCES $AUREOFACIENS)
AUTHOR J.SEVCIK,E.J.DODSON,G.G.DODSON
CRYST1 64.900 78.320 38.790 90.00 90.00 90.00 P 21 21 21 8
CONTNT H,K,L,S,FOBS,SIGMA(FOBS)
FORMAT (2(I3,2I4,2F7.0,F6.0,9X))
COORDS 2SAR
REMARK 1 TWO REFLECTIONS PER RECORD.
REMARK 2 DMIN=1.85, DMAX=16.28
CHKSUM 1 MIN H=0,MAX H=34,MIN K=0,MAX K=41,MIN L=0,MAX L=20
CHKSUM 2 TOTAL NUMBER OF REFLECTIONS=17346
CHKSUM 3 TOTAL NUMBER OF REFLECTION RECORDS=8673
CHKSUM 4 SUM OF FOBS=0.235499E+07
0 0 3 60 9 16 0 0 4 106 307 25
0 0 5 166 23 20 0 0 6 239 657 52
0 0 7 326 0 38 0 0 8 425 511 40
. . . . . . . . . . . . . . . . . . . . . .
D. Example of simple formatted file of amplitudes:¶
In this case the assumption is that order of data is H,K,L,F,sig(F)
2 3 4 12.3 1.2
-2 -3 -4 11.4 1.1
. . . . . . . . . . . . .
or
2 3 4 12.3
-2 -3 -4 11.4
. . . . . . . . .
The length of file records must not exceed 80 characters. The format of the records is free, e.g. data must be separated by blancs (be careful - some PDB files do not satisfy this rule).
COMMAND (BATCH) FILE EXAMPLES¶
(see also Testing_program_MOLREP as tutorial)
BATCH file example of Cross Rotation and Translation functions:¶
# --------------------------------
molrep -f fobs.dat -m mm1.crd -i <<stop
# --------------------------------
#
_NP 8
_RAD 27
_ANISO C
_sim .1
_compl .5
stop
BATCH file example of Self Rotation function:¶
# --------------------------------
molrep -f fobs.dat -i <<stop
# --------------------------------
_RAD 27
stop
BATCH example with MTZ file:¶
# --------------------------------
molrep -f p1.mtz -m p1_pdb.pdb -i <<stop
# --------------------------------
_F FO
_SIGF SDFO
_NP 8
_ANISO C
_sim .1
_compl .5
stop
BATCH example with MTZ file¶
For searching in the electron density map for some model (standard Rotation Function will be used):
# --------------------------------
molrep -f p1.mtz -m mod.pdb -i <<stop
# --------------------------------
_F FO
_SIGF SDFO
_PH PH_FO
_NP 8
stop
BATCH file example of fitting two models:¶
# --------------------------------
molrep -m mod1.pdb -mx mod2.pdb -i <<stop
# --------------------------------
_PRF Y
stop
BATCH file example of dyad search:¶
# --------------------------------
molrep -f fobs.dat -m mod1.pdb -i <<stop
# --------------------------------
_dyad y
_axis 0,10
_dist 0,300,300
_NPT 3
_NPTD 3
stop
BATCH file example of dimer search:¶
# --------------------------------
molrep -f fobs.dat -m mod1.pdb -i <<stop
# --------------------------------
_dyad y
_axis 180,10
_dist 0,300,1
_NPT 3
_NPTD 3
stop
BATCH file example dimer search for Self-RF orientations:¶
# --------------------------------
molrep -f fobs.dat -m mod1.pdb -i <<stop
# --------------------------------
_dyad y
_axis 180,10
_dist 0,300,1
_NSRF 20
_NPT 3
_NPTD 3
stop
BATCH file example of using file of sequence¶
# --------------------------------
molrep -f mtz.mtz -m 1hpg.pdb -s new.seq -i <<stop
# --------------------------------
_F FP
_SIGF SIGFP
_NP 8
_NMON 2
_sim .1
_compl .5
stop
How to define NCS¶
Program supports the point group symmetry.
NCS_ID |
Point group description. |
N00 |
Point group is N. For example Point group is 7, NCS_ID is 700. Standard orientation: Nfold axis along Z. |
N20 |
Point group is N2. For example Point group is 72, NCS_ID is 720. Standard orientation: Nfold axis along Z, twofold axis along X. |
N22 |
Point group is N22. For example Point group is 422, NCS_ID is 422. Standard orientation: Nfold axis along Z, twofold axis along X. |
230 |
Point group is 23. Standard orientation: twofold axis along Z, another twofold axis along X. |
432 |
Point group is 432. Standard orientation: fourfold axis along Z, another fourfold axis along X. |
532 |
Point group is 532. Standard orientation: fivefold axis along Z, projection closest (to Z axis) threefold axis in plan XY along X. |
Polar angles theta, phi, chi define the standard system orientation in the cell. Theta, phi - polar coordinates of Z standard axis. Chi - angle of rotation around theta-phi-axis (Z standard axis) which bring X axis to standard X axis.
cx,cy,cz (fract.units) define the position of group centre in the cell.
It is possible to define NCS parameters using keywords or in input PDB file.
Definition by keywords¶
Input PDB file must contain only one molecule.Use keywords:
Definition in input PDB file¶
First (reference) molecule must be started with line (free format):
#MOLECULE NCS_ID theta phi chi cx cy cz
Other molecules must be started with line:
#MOLECULE Nmol theta phi chi
where:
Nmol - molecule number.
theta phi chi - Polar angles of rotation from first molecule to current one.
For example: point group is 3.
HEADER HYDROLASE (ENDORIBONUCLEASE) CRYST1 64.900 78.320 38.790 90.00 90.00 ... #MOLECULE 300 0 0 0 .5 .5 .5 #DOMAIN 1 ATOM 1 N ASP A 1 45.161 12.836 ... ATOM 2 CA ASP A 1 45.220 12.435 ... ... ATOM 745 SG CYS A 96 58.398 6.673 ... ATOM 746 O CYS A 96 62.238 7.178 ... #DOMAIN 2 ATOM 747 N PHE A 97 44.487 11.386 ... ATOM 748 CA PHE A 97 44.559 11.129 ... ... ATOM 945 C VAL A 196 58.398 6.673 ... ATOM 946 O VAL A 196 62.238 7.178 ... #DOMAIN 1 ATOM 947 N ASP A 197 44.487 11.386 ... ATOM 948 CA ASP A 197 44.559 11.129 ... ... #MOLECULE 2 0 0 120 #DOMAIN 1 ATOM 1 N ASP A 1 45.161 12.836 ... ATOM 2 CA ASP A 1 45.220 12.435 ... ... ATOM 745 SG CYS A 96 58.398 6.673 ... ATOM 746 O CYS A 96 62.238 7.178 ... #DOMAIN 2 ATOM 747 N PHE A 97 44.487 11.386 ... ATOM 748 CA PHE A 97 44.559 11.129 ... ... ATOM 945 C VAL A 196 58.398 6.673 ... ATOM 946 O VAL A 196 62.238 7.178 ... #DOMAIN 1 ATOM 947 N ASP A 197 44.487 11.386 ... ATOM 948 CA ASP A 197 44.559 11.129 ... ... #MOLECULE 3 0 0 240 #DOMAIN 1 ATOM 1 N ASP A 1 45.161 12.836 ... ATOM 2 CA ASP A 1 45.220 12.435 ... ... ATOM 745 SG CYS A 96 58.398 6.673 ... ATOM 746 O CYS A 96 62.238 7.178 ... #DOMAIN 2 ATOM 747 N PHE A 97 44.487 11.386 ... ATOM 748 CA PHE A 97 44.559 11.129 ... ... ATOM 945 C VAL A 196 58.398 6.673 ... ATOM 946 O VAL A 196 62.238 7.178 ... #DOMAIN 1 ATOM 947 N ASP A 197 44.487 11.386 ... ATOM 948 CA ASP A 197 44.559 11.129 ... ...
Alternative way is to use only first molrecule (with NCS parameters in the file) and generate complete model automaticly. In pure RB refinement use keyword DOM = ‘C’. For fitting model into map (i.e. SAPTF+PRF+PTF use keyword NCS = 1).
How to redirect output and scratch files¶
In command string you can use options: “-po” and “-ps”.
For example:
Usual MR with sequence and redirect output and scratch files
molrep -f file.mtz -m model.pdb -s file_seq -po out/ -ps scr/
Convention for rotation
Rotation by Euleran angles Alpha, Beta, Gamma:
- euleran angles1. A( Z ) - alpha around axis Z
B( Y’) - beta around new axis Y
G( Z’) - gamma around new axis Z
Rotation by Polar angles Theta, Phi, Chi:
polar coordinates Theta, Phi of rotate axis:
Theta - angle between rotate axis and Z Phi - angle in plan XY between X and projection rotate axis
Chi - rotation angle arount rotate axis
Convention for Orthonormal coordinate system¶
Orthonormal axes are defined to have:
A parallel to X , Cstar parallel to Z