Macromolecular structure determination

Submitted by

-Chainika Das (10110009)

-Jha Ashutosh (10110025)

Objective To identify the structure of the given protein

sequence:

>gi|1336632|gb|AAB18304.1| 2,3-dihydro-2,3-dihydroxybiphenyl-2, 3-dehydrogenase [Comamonas testosteroni]

MKLTGEVALITGGASGLGRALVDRFVAEGARVAVLDKSAERLRELEVAHGGNAVGVVGDVRSLQDQKRAAERCLAAFGKIDTLIPNAGIWDYSTALADLPEDKIDAAFDDIFHVNVKGYIHAVKACLPALVSSRGSVVFTISNAGFYPNGGGPLYTATKHAVVGLVRQMAFELAPHVRVNGVAPGGMNTDLRGPSSLGLSEQSISSVPLADMLKSVLPIGRMPALEEYTGAYVFFATRGDSLPATGALLNYDGGMGVRGFLTAAGGADLPEKLNINREGQE

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`Target MTZ

&

Sequence

Target

Details

TemplateSearch

Model

Preparation

Molecular Replacement

& Refinement

Check scores

and exit or

select the next

model

Overall Process

Steps to follow(when search

model is given)

Given mtz, seq & pdb

MOLREP & Refinement (obtain score,

contrast)

WINCOOT (mutation, translation ,

validation, save as Refmac)

Model preparation

Refinement (check for R factor and R

free)

If search model is not available

Pblast - database

Similarity known structure is obtained by

using already present sequence.

1. Blast

Choose the hit with sequence

identity more than 25%

2. Matthew’s Coefficient

3. Molrep

-Score

-contrast

4. Refmac

-R factor

-R free

5. Wincoot

-Mutations

-Rotamers

-Ramachandranplot

6. Pymol

Active site

Procedure

MOLREP

REFMAC

Wincoot

CCP4

Scope: covers data processing through to

refinement and validation

Molecular Replacement programs:

Molrep

Phaser

Create New

Directory

Matthews Coefficient

Water must be approx 50 %

Molecular Replacement Molrep is program for automated molecular replacement in

the case where a homologous structure has already been

identified.

The program will attempt to find the number of molecules

expected in the asymmetric unit as entered by the user.

A PDB file for the best solution is output.

Additional options

Self rotation function

Search for model in a map

Alignment only

Can perform individual steps in more difficult cases

MOLREP

3 inputs:

o Mtz file – experiment data

o Pdb file- search model

o Use Sequence

Output is .pdb (to be input in refmac) file

- positioned search model

-input for the next stage (restrained

refinement)

Steps

Log file

Steps

Score: product of correlation coefficient of intensity and maximum value of packing fraction.Contrast: Ratio of Top score to mean score. Should be >2.5

Steps

Refmac

The search model obtained from MOLREP

is passed to Refmac for refinement.

The change in the Rfree & R factor value

during refinement is used as a measure to

check how good the resulting model is.

Steps

Log file

Steps

1. Free-R: R-factor is calculated for a test-set of reflections that is never

included in refinement.

Difference between R and R-free is smaller for higher resolution and well-refined structures

Refinement is carried out in repetitive cycles till R-factor converges to a low value with appropriate geometry of the atomic model.

This value is 5% from the overall data.

2. R-factor:

It is a measure of how well the refined structure predicts the observed value

R =∑ |(Fobs)|-|(Fcalc)| ∑|(fobs)|

It should be 15-20% for macromolecules.

Wincoot

It is used for manipulation and

visualization of the Refmac5 pdb file.

With the help of overlapping electron

density the amino acids are identified and

fit in the structure.

The red and green regions are removed.

Steps

Steps

Steps

The red density indicates incorrect position

of proteins while the green density

indicates the scope of placing the

proteins.

On the overlap of blue and green density

side chains should be placed.

Steps

Steps

Validation

Ramachandran Plot

Rotamer analysis

Steps

If >80%, structure is correct.

6 outliers

Ramachandran Plot

Steps

Change residues Phi & Psi to remove outliers

Steps

Steps

Steps

Steps

Back To CCP4- Refmac

Steps

Input new

pdb file saved

from Coot

Steps

Steps

Rotamer Analysis

Red Bars Show Poor Rotamers

Steps

Poor rotamer is removed

Steps

Steps

Fill Incomplete areas, use Sequence file to know the missing sequence

Steps

Steps

Steps

Final R factor is less than the initial.

PyMOL

It is an open-source, user-sponsored,

molecular visualization system.

It is well suited to producing high quality

3D images of small molecules and

biological macromolecules such as

proteins.