Refinement procedure
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![Page 1: Refinement procedure](https://reader038.fdocuments.us/reader038/viewer/2022102707/56812fa4550346895d952250/html5/thumbnails/1.jpg)
Refinement procedure
Copy your best coordinate file to “prok-native-r1.pdb”:
cp yourname-coot-99.pdb prok-native-r1.pdb
Start refinement
phenix.refine prok-native-r1.pdb prok-native-mcollazo.mtz
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obs
Structure Refinement Schematic
Reciprocal Space
Real Space
|Fobs-native |
|Fobs-PCMBS |
|Fobs-EuCl3 |
Fit Map
FT (Coot) FT (Phenix)
|Fcalc |in
|Fobs-Fcalc|
|Fobs||Fcalc|out
Manual Refinement
Build atoms to
FT (Coot)
2Fobs-Fcalc
map
Fit |Fobs|
Move atoms to
calc
Manual Refinement
FT (Phenix)
|Fcalc |in
Automatic Refinement
experimental map
Fobs-Fcalc
map
coordinates(prok-native-r1_refine_001.pdb)
coordinates(prok-native-r2.pdb)
coordinates(prok-native-r1.pdb)
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Get a sorted list of Fobs-Fcalc peaks
Ramachandran plotKleywegt plotIncorrect Chiral VolumesUnmodeled BlobsDifference Map peaksCheck/Delete WatersGeometry AnalysisPeptide Omega AnalysisRotamer AnalysisDensity Fit AnalysisProbe ClashesNCS differencesPukka PuckersAlignment vs. PIR
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Fobs-Fcalc reveals errors in model
Positive densityNegative density
Real Space Refine and dragOr Autofit Rotamer
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Fobs-Fcalc reveals errors in model
Real Space Refine and dragOr Autofit Rotamer
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water
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water
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Other solvent
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Other solvent
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Goals for Today
• Automated Refinement of ProK – Phenix– Rwork and Rfree for your model.
• Manual Refinement of ProK – correct errors with Coot
• Automated Refinement of ProK – Phenix– Rwork and Rfree for your model.
• Validate ProK model (web server)• Awards• Refine ProK-PCMBS complex • Go forth wielding the tools of X-ray crystallography and
discover the secrets of other biological macromolecules.
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REAL vs RECIPROCAL
• Real Space • Manual• Local• Improvement in the
model is limited by the quality of the phases
• Large radius of convergence
• Reciprocal Space• Automatic• Global• Improved phases will lead
to improved maps and improved interpretability and improved model.
• Small radius of convergence
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Radius of convergence
• Manual adjustments improve radius of convergence
Rupp
Torsion angle C-C
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Reciprocal Space Target function: Edata (R-factor)
Move atoms to minimize the R-factor. Minimize the discrepancy between Fobs and Fcalc.
Specifically, minimize EEdata=w(Fobs-Fcalc)2 Over all hkl.
Past--We used least squares minimization to refine.
Now--Maximum likelihood allows for non-random error model. Given this model, what is the probability that the given set of data would be observed.
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Importance of supplementing the Data to Parameter Ratio
in crystallographic refinement.
PARAMETERSEach atom has 4 parameters (variables) to refine:
x coordinatey coordinatez coordinateB factor
In proteinase K there are approximately 2000 atoms to refine.
This corresponds to
2000*4= 8000 variables.
At 1.7 A resolution we have 25,000 observations. About 3 observations per variable. The reliability of the model is still questionable.
Adding stereochemical restraints is equivalent to adding observations
DATAAt 2.5 A resolution we have 8400 observations (data points) (Fobs).
When # of observations= # of variablesA perfect fit can be obtained irrespective of the accuracy of the model.
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Automated Refinement(distinct from manual building)
Two TERMS:
Etotal = Edata(wdata)+ Estereochemistry
Edata describes the difference between observed and calculated data.
wdata is a weight chosen to balance the gradients arising from the two
terms.
Estereochemistry comprises empirical information about chemical
interactions between atoms in the model. It is a function of all atomic positions andincludes information about both covalent and non-bonded interactions.
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Estereochemistry (Geometry)
–BOND LENGTHS & ANGLES have ideal values. Engh & Huber dictionary.CHIRALITY of -carbons–PLANARITY of peptide bonds and aromatic side chains –NONBONDED CONTACTS -two atoms cannot occupy the same space at the same time-TORSION ANGLE PREFERENCES side chains have preferred rotamers.–some values of and are forbidden. -Ramachandran. Not restrained- used for validation.
e
loop__chem_comp_bond.comp_id_chem_comp_bond.atom_id_1_chem_comp_bond.atom_id_2_chem_comp_bond.type_chem_comp_bond.value_dist_chem_comp_bond.value_dist_esd ALA N CA single 1.458 0.019 ALA CA CB single 1.521 0.033 ALA CA C single 1.525 0.021 ALA C O double 1.231 0.020
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Jeopardy clue:
The appearance of the atomic model when stereochemical restraints are not included in
crystallographic refinement.
What is spaghetti, Alex?
Etotal =Estereochemistry + wdataEdata
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restrained not restrained
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2nd Jeopardy clue:
The value of the R-factor resulting when stereochemical restraints are not included in
crystallographic refinement.
What is zero, Alex?
Etotal =Estereochemistry + wdataEdata
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An atomic model should be validated by several unbiased indicators
Low RMS deviations in bond lengths and angles does not guarantee a correct structureRfree is an unbiased indicator of the
discrepancy between the model and the data. The data used in this R-factor calculation were not used in determining atomic shifts in the refinement process.
Ramachandran plot is unbiased because phi and psi torsion angles are not restrained in the refinement process.
The need for Cross-Validation
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Stop Here
• Now, use COOT to correct errors in Phenix refined model: – prok-native_refine_001.pdb– Spend 15 minutes
• Run Phenix after COOT
• Resume discussion on structure validation while Phenix is running.
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O
NH
BACKBONE AMIDE
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N O
Asn
H
H
O
NH
BACKBONE AMIDE
2.8 Å
BAD
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NO
H
H
O
NH
BACKBONE AMIDE
2.8 Å
Asn
GOOD
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ERRAT examines distances between non-bonded atoms. Reports the deviations of C-C, C-N, C-O, N-N, N-O, O-O distances from distributions characteristic of reliable structures.
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Verify 3D plot Indicates if the sequence has been improperly threaded through the density. It measures the compatibility of a model with its sequence. Evaluate for each residue in the structure: (1) Surface area buried (2) Fraction of side-chain area covered by polar atoms (3) Local secondary structure and compare to ideal library values for each amino acid type.
Report the fraction of residues with score greater than 0.2
Backwards trace
Correct trace
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Submit coordinates to SAVS server
• Google for “UCLA SAVES”
• Continue with discussion on solving the ProK-PCMBS complex structure.
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F
Plan for today: Solve structure of ProK-PCMBS complex
S
ProKactive siteCys74
PMB: p-chloromercuribenzoylsulfonate
HCl
HgSO3
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The beauty of isomorphism
protein a (Å) b (Å) c (Å)
ProK 67.9 67.9 101.8 90° 90° 90°
ProK+PCMBS 67.9 67.9 102.5 90° 90° 90°
Riso=15.2%What is maximum possible Riso?What is minimum possible Riso?
• Initial phases: phases from native proteinase K structure calc ProK. • Fobs amplitudes: Use |FProk-PCMBS| data measured earlier in the course.
Why don’t we have to use Heavy atoms?Why don’t we have to use Molecular Replacement?
x,y,z=1/V*|Fobs|e-2i(hx+ky+lz-calc
)
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Fo-Fc Difference Fourier map
x,y,z=1/V*|Fobs-Fcalc|e-2i(hx+ky+lz-calc
)
•Here, Fobs will correspond to the Proteinase K-PMSF complex.•Fcalc will correspond to the model of Proteinase K by itself after a few cycles of automated refinement.•Positive electron density will correspond to features present in the PMSF complex that are not in the native structure.•Negative electron density will correspond to features present in the native structure that should be removed in the inhibitor complex.•After model building, do more automated refinement and then validate.
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4 Key Concepts
• When to use isomorphous difference Fourier to solve the phase problem.
• How to interpret an Fo-Fc Difference Fourier map.
• Expected values of RMS deviation from ideal geometry
• methods of cross-validation
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Validate protein structure by Running SAVES server
grep -v hex prok-native_refine_001.pdb >prok-pmsf.pdb
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Name _______________________
Refinement statistics Proteinase K native
Proteinase K-PMSF
Resolution
Molecules in asymmetric unit 1
Solvent content (%) 36.3
Matthews coefficient (Å3/Da) 1.9
Number of reflections used
Rwork
Rfree
RMSD Bond lengths
RMSD Bond angles
Ramachandran plot: favored
Ramachandran plot: allowed
Ramachandran plot: generously allowed
Ramachandran plot: outliers
Number of atoms: protein
Number of atoms: solvent
Errat overall quality factor
percentage with Verify3D score>0.2
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R R
Cis vs. Trans peptide
C
C
O
N
C
peptide plane
RR
C
O
N
Cpeptide plane
C
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Cis OK with glycine or proline
RH
C
C
O
N
C
peptide plane
R
C
C
O
N
C
peptide plane
Steric hindrance equivalentfor cis or trans.
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Steric hindrance equivalentfor cis or trans proline
.
R
C
C N
C
peptide planeO
R
C
C N
peptide planeO
C
C
C
C C
CC