Combined MR-SAD using CCP4i - Revision history

Randy: Give link for tutorials files.

2018-11-05T10:53:19Z

Give link for tutorials files.

Randy: Change to use parrot HL coeffs in both ARP/wARP runs.

2018-11-05T10:31:58Z

Change to use parrot HL coeffs in both ARP/wARP runs.

Randy: Fix percent identity

2018-11-05T10:08:47Z

Fix percent identity

Randy: Update ARP/wARP instructions for version 8.0

2018-11-04T16:33:12Z

Update ARP/wARP instructions for version 8.0

Randy: Suggest using Rfree flags in ARP/wARP.

2016-04-06T09:56:17Z

Suggest using Rfree flags in ARP/wARP.

Randy: Suggest using sculptor on the goat model.

2016-04-05T18:40:52Z

Suggest using sculptor on the goat model.

Randy: Bring up to date with CCP4 6.3, using Parrot instead of DM and ARP/wARP Classic instead of Expert System

2012-11-23T19:10:40Z

Bring up to date with CCP4 6.3, using Parrot instead of DM and ARP/wARP Classic instead of Expert System

WikiSysop: add category

2009-07-13T09:49:47Z

add category

WikiSysop: Protected "Combined MR-SAD using CCP4i" ([edit=sysop] (indefinite) [move=sysop] (indefinite)) [cascading]

2009-07-09T09:41:31Z

Protected "Combined MR-SAD using CCP4i" ([edit=sysop] (indefinite) [move=sysop] (indefinite)) [cascading]

WikiSysop: Initialization from almanac

2009-06-30T12:24:48Z

Initialization from almanac

New page

All files for this tutorial are distributed from the Phaser web page
Reflection data: lyso2001_scala1.mtz
Lactalbumin model: 1fkq_prot.pdb
Sequence file: hewl.pir

This tutorial illustrates a common molecular replacement/experimental phasing scenario, when refinement is hindered by very strong model bias, but there is some experimental phasing signal available.

Goat α-lactalbumin is 45% identical to hen egg-white lysozyme. Although it is possible to solve lysozyme using α-lactalbumin as a model, it is very difficult to refine the structure, partly because of model bias. Unfortunately, low solvent content of this crystal form limits the ability of density modification to remove the bias. However, one can use anomalous scattering from intrinsic sulfur atoms to improve phases dramatically. It is noteworthy that the anomalous signal from the sulfur atoms is not sufficient for ab initio phasing (it is not possible to locate the anomalous scatterers from the data alone).

# Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary.
# For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do a quick solvent flattening with DM using AUTO cycles (input Hendrickson-Lattman coefficients, and set PHIO=PHIB and FOMO=FOM). The best results are obtained if DM starts with the map produced by Phaser instead of a simple FOM-weighted map. In CCP4 6.1, you can check the box labeled "Input starting map coefficients" and enter FDM=FWT and PHIDM=PHWT. For older versions of CCP4, you have to start the job with "Run&View Com File", edit the LABIN keyword to include the following: FDM=FWT PHIDM=PHWT, and then run the job.
# Start up ARP/wARP Expert System in "automated model building starting from experimental phases" mode. Start from the DM phases, and include HL coefficients for phase restraints (use the ones from Phaser).
# Now add the S-SAD phase information. Bring up the GUI for Phaser in the Experimental Phasing module
# All the yellow boxes need to be filled in.
#* Set "Mode for experimental phasing" to SAD with molecular replacement partial structure.
#* Set "LLG-map calculation atom type" to S.
#* Under the "Define atoms" heading, set "Partial structure" to the molecular replacement solution (output PDB-file) you have obtained in step 1.
# Run Phaser after you entered all the information.
# Solvent flatten with DM using the same protocol as in step 2.
# Run ARP/wARP Expert System using the same protocol as in step 3.
# How many anomalous scatterers has Phaser found? Check them against the model and guess what they may be! Why is it not important to specify the exact element type in this case?
# If you did not know the correct space group, would you have to run Phaser twice?
# Compare the two ARP/wARP runs! Which one has built more residues?

@@ Line 10: / Line 10: @@
 # Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary. Better results will be achieved if you run sculptor to modify the model on the basis of the supplied sequence alignment.
-# For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do a quick solvent flattening with Parrot (choose "Use PHI/FOM instead of HL coefficients" because the MTZ file produced after MR doesn't include Hendrickson-Lattman coefficients; set PHI=PHIC and FOM=FOM; select "Use map coefficients", then set F=FWT and PHI=PHWT).
+# For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do solvent flattening with Parrot (choose "Use PHI/FOM instead of HL coefficients" because the MTZ file produced after MR doesn't include Hendrickson-Lattman coefficients; set PHI=PHIC and FOM=FOM; select "Use map coefficients", then set F=FWT and PHI=PHWT).
-# Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the Required parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence file, and note there are 129 residues in lysozyme. Select "use" the Free R flag. To save time, do 3 cycles of autobuilding instead of 10.
+# Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the Required parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. In the same section, select "Phased ML" for the Refmac target function, and check that the GUI has automatically selected the parrot Hendrickson-Lattman coefficients (parrot.ABCD.A, etc.). Select the sequence file, and note there are 129 residues in lysozyme. Select "use" the Free R flag. To save time, do 3 cycles of autobuilding instead of 10.
 # Now add the S-SAD phase information. Bring up the GUI for Phaser SAD pipeline in the Automated Search & Phasing section of the Experimental Phasing module
 # All the yellow boxes need to be filled in.
@@ Line 20: / Line 20: @@
 # Run Phaser after you entered all the information.
 # Solvent flatten with Parrot using a similar protocol to step 2.  However, you should *not* choose "Use PHI/FOM instead of HL coefficients".  Choose the HLanomA/B/C/D coefficients because these describe the phase information obtained only from the anomalous scattering information and not from the molecular replacement model; using HLA/B/C/D would include the model phase information, which would bias maps from subsequent cycles of phase improvement to look like the model.
-# Run ARP/wARP using a similar protocol as in step 3, except you should open the Refmac parameters folder and choose the option to include HL phase restraints.  In the MTZ data section, choose the HLanomA/B/C/D coefficients.
+# Run ARP/wARP using the same protocol as in step 3.
 # How many anomalous scatterers has Phaser found? Check them against the model and guess what they may be! Why is it not important to specify the exact element type in this case?
 # If you did not know the correct space group (from the MR step), would you have to run Phaser SAD-phasing twice?

@@ Line 7: / Line 7: @@
 This tutorial illustrates a common molecular replacement/experimental phasing scenario, when refinement is hindered by very strong model bias, but there is some experimental phasing signal available.
-Goat α-lactalbumin is 45% identical to hen egg-white lysozyme. Although it is possible to solve lysozyme using α-lactalbumin as a model, it is very difficult to refine the structure, partly because of model bias. Unfortunately, low solvent content of this crystal form limits the ability of density modification to remove the bias. However, one can use anomalous scattering from intrinsic sulfur atoms to improve phases dramatically. It is noteworthy that the anomalous signal from the sulfur atoms is not sufficient for ab initio phasing (it is not possible to locate the anomalous scatterers from the data alone).
+Goat α-lactalbumin is 42% identical to hen egg-white lysozyme. Although it is possible to solve lysozyme using α-lactalbumin as a model, it is very difficult to refine the structure, partly because of model bias. Unfortunately, low solvent content of this crystal form limits the ability of density modification to remove the bias. However, one can use anomalous scattering from intrinsic sulfur atoms to improve phases dramatically. It is noteworthy that the anomalous signal from the sulfur atoms is not sufficient for ab initio phasing (it is not possible to locate the anomalous scatterers from the data alone).
 # Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary. Better results will be achieved if you run sculptor to modify the model on the basis of the supplied sequence alignment.

@@ Line 11: / Line 11: @@
 # Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary. Better results will be achieved if you run sculptor to modify the model on the basis of the supplied sequence alignment.
 # For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do a quick solvent flattening with Parrot (choose "Use PHI/FOM instead of HL coefficients" because the MTZ file produced after MR doesn't include Hendrickson-Lattman coefficients; set PHI=PHIC and FOM=FOM; select "Use map coefficients", then set F=FWT and PHI=PHWT).
-# Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the ARP/wARP flow parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence file, and note there are 129 residues in lysozyme. Select "use" the Free R flag. To save time, do 3 cycles of autobuilding instead of 10.
+# Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the Required parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence file, and note there are 129 residues in lysozyme. Select "use" the Free R flag. To save time, do 3 cycles of autobuilding instead of 10.
 # Now add the S-SAD phase information. Bring up the GUI for Phaser SAD pipeline in the Automated Search & Phasing section of the Experimental Phasing module
 # All the yellow boxes need to be filled in.

@@ Line 11: / Line 11: @@
 # Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary. Better results will be achieved if you run sculptor to modify the model on the basis of the supplied sequence alignment.
 # For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do a quick solvent flattening with Parrot (choose "Use PHI/FOM instead of HL coefficients" because the MTZ file produced after MR doesn't include Hendrickson-Lattman coefficients; set PHI=PHIC and FOM=FOM; select "Use map coefficients", then set F=FWT and PHI=PHWT).
-# Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the ARP/wARP flow parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence file, and note there are 129 residues in lysozyme.  To save time, do 3 cycles of autobuilding instead of 10.
+# Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the ARP/wARP flow parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence file, and note there are 129 residues in lysozyme. Select "use" the Free R flag. To save time, do 3 cycles of autobuilding instead of 10.
 # Now add the S-SAD phase information. Bring up the GUI for Phaser SAD pipeline in the Automated Search & Phasing section of the Experimental Phasing module
 # All the yellow boxes need to be filled in.

@@ Line 3: / Line 3: @@
   Lactalbumin model: 1fkq_prot.pdb
   Sequence file: hewl.pir
 This tutorial illustrates a common molecular replacement/experimental phasing scenario, when refinement is hindered by very strong model bias, but there is some experimental phasing signal available.
@@ Line 8: / Line 9: @@
 Goat α-lactalbumin is 45% identical to hen egg-white lysozyme. Although it is possible to solve lysozyme using α-lactalbumin as a model, it is very difficult to refine the structure, partly because of model bias. Unfortunately, low solvent content of this crystal form limits the ability of density modification to remove the bias. However, one can use anomalous scattering from intrinsic sulfur atoms to improve phases dramatically. It is noteworthy that the anomalous signal from the sulfur atoms is not sufficient for ab initio phasing (it is not possible to locate the anomalous scatterers from the data alone).
-# Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary.
+# Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary. Better results will be achieved if you run sculptor to modify the model on the basis of the supplied sequence alignment.
 # For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do a quick solvent flattening with Parrot (choose "Use PHI/FOM instead of HL coefficients" because the MTZ file produced after MR doesn't include Hendrickson-Lattman coefficients; set PHI=PHIC and FOM=FOM; select "Use map coefficients", then set F=FWT and PHI=PHWT).
 # Start up ARP/wARP Classic in "automated model building starting from experimental phases" mode. Select the MTZ file from Parrot.  To start from the Parrot map, select "Use a different (pre-weighted) Fobs for initial map calculation" under the ARP/wARP flow parameters folder, then set FBEST=parrot.F_phi.F, PHIB=parrot.F_phi.phi and FOM=Unassigned. Select the sequence file, and note there are 129 residues in lysozyme.  To save time, do 3 cycles of autobuilding instead of 10.
@@ Line 16: / Line 17: @@
 #* Uncheck the Parrot and Buccaneer steps of the pipeline (to allow control for better comparison with the MR model alone).
 #* Set LLG-map calculation atom type to S.
-#* Under the "Define atoms" heading, set "Partial structure" to the molecular replacement solution (output PDB-file) you have obtained in step 1.
+#* Under the "Define atoms" heading, set "Partial structure" to the molecular replacement solution (output PDB-file) you have obtained in step 1. You can enter either the refined estimated RMSD from the molecular replacement step or the sequence identity.
 # Run Phaser after you entered all the information.
 # Solvent flatten with Parrot using a similar protocol to step 2.  However, you should *not* choose "Use PHI/FOM instead of HL coefficients".  Choose the HLanomA/B/C/D coefficients because these describe the phase information obtained only from the anomalous scattering information and not from the molecular replacement model; using HLA/B/C/D would include the model phase information, which would bias maps from subsequent cycles of phase improvement to look like the model.

@@ Line 1: / Line 1: @@
-All files for this tutorial are distributed from the Phaser web page
+All files for this tutorial are found [http://www.phaser.cimr.cam.ac.uk/index.php/Tutorials here]
   Reflection data: lyso2001_scala1.mtz
   Lactalbumin model: 1fkq_prot.pdb

← Older revision		Revision as of 09:49, 13 July 2009
Line 22:		Line 22:
	# If you did not know the correct space group, would you have to run Phaser twice?		# If you did not know the correct space group, would you have to run Phaser twice?
	# Compare the two ARP/wARP runs! Which one has built more residues?		# Compare the two ARP/wARP runs! Which one has built more residues?
		+
		+
		+	[[Category:Tutorial]]

← Older revision	Revision as of 09:41, 9 July 2009
(No difference)