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Lysozyme Crystals – Week 3
Working on Refinement/Model Building
Files
•
•
Create directory in which you will perform all your work.
Obtain PDB file containing molecular replacement solution from Moodle and place in
folder. Likewise obtain the data (“mtz”) file and place it similarly.
Starting Refmac5
•
Start CCP4 and click on Directories Button on upper right of window. Name a project and
then “browse” to find you’re newly made folder. Make sure your “Current Project” is the
one listed and “Apply and Exit”.
•
From the left hand list of options, choose “Refinement” and from the new menu, select
“Refmac5”. A pop-up form will appear.
Rigid body refinement
•
Job title is optional
•
Do [rigid body refinement] with [no prior phase information] and [no] twin refinement.
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With MTZ in, your [Project] file should be selected. Click on browse and pick the MTZ file.
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The MTZ out should also go to [Project]. I would name it “rigid.mtz”.
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Likewise with the PDB in and PDB out (name it rigid.pdb).
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Click on the button next to refinement parameters.
Change resolution range. Keep the low res #, but select 3.5 Å for the high res.
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If all looks good, click on the “Run” button and select “Run Now”.
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Monitor Rwork/Rfree in log file. To do so, return to the main CCP4 window. Click on the
“Refmac5” line in the jobs list. When it says “Finished” go to “View Files From Job” and
ask to see “View Log File”. Scroll to the bottom to view the change in R-factors.
XYZB refinement
•
Return to Refmac5 window. Change [rigid body refinement] to [restrained refinement].
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Retain the original MTZ in file. Change the name of the MTZ out to refine1.mtz.
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Change PDB in to rigid.pdb. Change the PDB out to refine.pdb.
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Under refinement options
Select desired high resolution limit (usually highest resolution of data set).
•
You’re ready to run again. Again monitor Rwork/Rfree and now start monitoring rmsBond and
rmsAngl(es) as well in the Log File.
Coot: Rebuild One
•
Start software. Load coordinate file (File/Open Coordinates) using the name of the output
PDB file from XYZB body refinement. Load the maps (File/Auto Open MTZ…) from the
mtz file output during XYZB body refinement.
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Navigate the model using the (Draw/Go To Atom…) option. Alternatively, if you middle
click on an atom, the screen will center on that atom.
•
•
•
Interpret the maps. Residues should be coated in blue mesh (2Fo – Fc map). Where you see
green (positive Fo-Fc) there is something missing. Where you see red (negative Fo-Fc) too
much is present.
Fixing sequence. Navigate to residues where HEWL sequence varies from the model. Use
the (Mutate & AutoFit) button to make changes. Click on residue to be changed and then
on residue type you want.
When finished, save coordinates (File/Save Coordinates) and exit.
XYZB Refinement: Two
•
Identical to the previous round. But substitute PDB in with your output from Coot and
alter MTZ out and PDB out to refine2.***.
Coot: Rebuild Two
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Open software and load coordinates (refine2.pdb) and auto open MTZ (refine2.mtz). In
addition, open the anomalous map using “Open Map” command.
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You may inspect the model residue by residue looking for poorly fit side chains.
•
Alternately you can look for explicit regions of difficulty.
(Validate/Density Fit Analysis) color codes residues by quality of fit to the map.
(Validate/Unmodeled Blobs) will look for poorly modeled regions in the map.
•
You may also add waters by hand, looking for spheres of unmodeled density (add water
button) or you may do it automatically (Calculate/Other Modeling Tools/Find Waters…).
Use Map 2 (DELFWT) and go to 3.5 or 4 sigma. I would keep spacing above 2.4 and below
3.2 Å. Add waters to the “model that masks the map”.
•
If you add waters automatically, review each one. This is easiest to do by clicking on them in
the (Validate/Density Fit Analysis) window. If the water is in a non-spherical blob of
density, beware. Delete any that have no map density as well (use trash can icon).
From here on out you continue to alternate refinement and rebuild until you are tired or there is no
further improvement in R/Rfree. Always end with a round of refinement.
Validation
•
You will of course cite R/Rfree for your final model as well as the RMS differences for your
bond lengths and angles from the reference library (these appear in the text header at the
beginning of the PDB file). In addition, use the Ramachandran plot option in Coot
(Validate/Ramachandran) to assess stereochemical quality.
Tuesday Data Set (298 K)
Overall
12.53
1.90
Low resolution limit
High resolution limit
Rmerge
Rmerge in top intensity bin
Total number of observations
Total number unique
Mean((I)/sd(I))
Completeness
Multiplicity
Anomalous completeness
Anomalous multiplicity
DelAnom correlation between half-sets
Mid-Slope of Anom Normal Probability
Average unit cell:
Space group: P43212
Average mosaicity:
79.22
79.22
InnerShell
12.53
6.01
OuterShell
2.00
1.90
0.056
0.036
107565
9671
27.0
97.6
11.1
0.038
3923
330
43.8
89.5
11.9
0.141
5150
1205
7.1
86.6
4.3
96.7
5.9
0.120
0.950
93.1
7.6
0.471
-
81.1
2.2
-0.063
-
37.65
90.00
90.00
90.00
0.69
Wednesday Data Set (100 K)
Overall
12.36
1.90
Low resolution limit
High resolution limit
Rmerge
Rmerge in top intensity bin
Total number of observations
Total number unique
Mean((I)/sd(I))
Completeness
Multiplicity
Anomalous completeness
Anomalous multiplicity
DelAnom correlation between half-sets
Mid-Slope of Anom Normal Probability
Average unit cell:
Space group: P43212
Average mosaicity:
77.90
0.73
77.90
37.09
InnerShell
12.36
6.01
OuterShell
2.00
1.90
0.054
0.036
102125
8915
31.3
94.4
11.5
0.044
3734
311
42.9
88.9
12.0
0.106
4906
978
10.0
73.5
5.0
92.7
6.2
0.102
0.980
92.7
7.7
0.497
-
65.4
2.6
-0.086
-
90.00
90.00
90.00
Experimental details:
X-ray data was collected from hen egg white lysozyme crystals grown from 0.1 M sodium acetate,
pH 4.6 and 1.2 M NaCl. The crystals were transferred sequentially the mother liquor containing 1
mM eosin and 10%, then 20%, then 30% glycerol. Data were collected at *** K on a Rigaku
Minimax system, incorporating a Saturn 724 CCD detector and Micromax-003 generator providing
copper Kα radiation. Data were integrated using Mosflm and scaled using Scala. A molecular
replacement solution was obtained using Molrep software. Rotation and translation solutions were
obtained from *** as an initial model which possesses *** % sequence identity to the hen egg white
enzyme.
A bibliography
Scala
Collaborative Computational Project, Number 4 (1994). The CCP4 Suite: Programs for Protein
Crystallography. Acta Cryst. D50, 760-763.
Mosflm
Leslie, A. G. W. (1992) Recent changes to the MOSFLM package for processing film and image
plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 26.
Refmac5
Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of Macromolecular
Structures by the Maximum-Likelihood Method. Acta Crystallographica, Section D Biological
Crystallography 54, 1285-1294.
Coot
Emsley, P., Lohkamp, B. and Scott, W. G. (2010) Features and Development of Coot. Acta
Crystallographica, Section D Biological Crystallography 66, 486-501.
Pheasant Chicken sequence alignment
Identities = 120/129 (93%), Positives = 123/129 (95%), Gaps = 0/129 (0%)
Pheas
2
HEWL
1
Pheas
62
HEWL
61
Pheas
122
HEWL
121
KVYGRCELAAAMKRMGLDNYRGYSLGNWVCAAKFESNFNTGATNRNTDGSTDYGILQINS
KV+GRCELAAAMKR GLDNYRGYSLGNWVCAAKFESNFNT ATNRNTDGSTDYGILQINS
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINS
RWWCNDGRTPGSKNLCHIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRKHCKGTDV
RWWCNDGRTPGS+NLC+IPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWR CKGTDV
RWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDV
NVWIRGCRL
WIRGCRL
QAWIRGCRL
130
129
61
60
121
120