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

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)

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

Fobs-Fcalc reveals errors in model

Positive densityNegative density

Real Space Refine and dragOr Autofit Rotamer

Fobs-Fcalc reveals errors in model

Real Space Refine and dragOr Autofit Rotamer

water

water

Other solvent

Other solvent

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.

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

Radius of convergence

• Manual adjustments improve radius of convergence

Rupp

Torsion angle C-C

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.

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.

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.

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

Jeopardy clue:

The appearance of the atomic model when stereochemical restraints are not included in

crystallographic refinement.

What is spaghetti, Alex?

Etotal =Estereochemistry + wdataEdata

restrained not restrained

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

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

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.

O

NH

BACKBONE AMIDE

N O

Asn

H

H

O

NH

BACKBONE AMIDE

2.8 Å

BAD

NO

H

H

O

NH

BACKBONE AMIDE

2.8 Å

Asn

GOOD

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.

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

Submit coordinates to SAVS server

• Google for “UCLA SAVES”

• Continue with discussion on solving the ProK-PCMBS complex structure.

F

Plan for today: Solve structure of ProK-PCMBS complex

S

ProKactive siteCys74

PMB: p-chloromercuribenzoylsulfonate

HCl

HgSO3

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

)

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.

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

Validate protein structure by Running SAVES server

grep -v hex prok-native_refine_001.pdb >prok-pmsf.pdb

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

R R

Cis vs. Trans peptide

C

C

O

N

C

peptide plane

RR

C

O

N

Cpeptide plane

C

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.

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