Electrostatic Potential (ESP)Measure polarizationElectron Map densityElectron distribution

Dipole MomentMeasure bond length/angle

Measure bond strength

Organic software for 3D model

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Modelling and 3D representation

Chemistry Database

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Spectroscopic Database

Click here down Swiss PDB

Modelling and 3D representation

Click here crystallography database.

✓ ✓

Click here NIST data

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Click here chem EdDL

Click here download chimera✓

Measure polarizationElectron Map densityElectron distribution

Electrostatic Potential (ESP)Dipole Moment

Measure bond length/angleMeasure bond strength

Organic software for 3D model

Click here download Rasmol

Click here download PyMolClick here download Jmol

Click here Chem EDDL

Click here chemical search.

Modelling and 3D representation

Quick Chemistry Database Check

Click here down Swiss PDB

Modelling and 3D representation

✓ ✓

Click here NIST data

✓Click here download Arguslab

Click here chemaxon quick chem check

Click here download Avagrado

Click here chem EdDL

Click here for Visualization/3D sources

Click here download Marvin Sketch

Click here quick chemical checkClick here quick chemical check

Type PDB code – 4HHBRight click – select Hetero Select - HETATM – HEM

4 Heme is display from 4 chains

Measure bond length/angleMeasure number H2 bonds

Measure bond strengthProtein 1, 2 , 3O structure

Presence of disulfide bondPresence alpha and beta pleated sheet

Click here J mol protein video

Chemical viewer 3D structure (Jmol)

Uses molecular modelling

1

J mol executable file

Measure distance

Select measure – distance for porphyrin ringMeasure ring size/distance Fe from planeSelect protein – by residue – HistidineMeasure and locate His F8 Measure and locate His E7

final heme – click here

J mol executable file

1

Type 4HHB into protein data bank

Look for ligand Heme

Model kit to design molecule

Click here deoxyhemoglobin chimera

22

3

4

3

4

final productAll histidine shown

Get structure fromPDB and MOL

Measure bond length/angleMeasure number H2 bonds

Measure bond strengthProtein 1, 2 , 3O structure

Presence of disulfide bondPresence alpha and beta pleated sheet

Protein Data BankProtein database key in - PDB 4HHB

Click here Chimera tutorial

1

2

Uses molecular modelling

1

2

Chemical viewer 3D structure (Chimera)

Download PDB text file

File – fetch by ID- 4HHB

Select – residue – HEMSelect – chain A – Action – Ribbon – HideSelect – chain B,C,D - Action – ribbon HideDisplay only ligand Heme

Tool- structural analysis - DistanceSelect 2 atom -by press control/shift/left click select 2 pointsTool – structure analysis – create to get distance

3

Check here 4HHB Chimera 1MBOSelect Histidine that are close to ringLocate His F8 and E7Make measurement

Click here download chimera

Tool – Sequence – choose sequence for 4 chainsIdentify amino acids of interest

4

Measure bond length/angleMeasure number H2 bonds

Measure bond strengthProtein 1, 2 , 3O structure

Presence of disulfide bondPresence alpha and beta pleated sheet

Organic software for 3D model (Pymol)

1 1

Click here - Protein Data BankProtein database key in - PDB 4 letter code

3

Click here download PyMol

Click here Pymol video tutorial

Click file – open your download pdb file from Protein Data bankGet to command term – Type fetch 4HHBH - Hide – S - Show cartoon – C – Type by ss

Select 4 Hem – Look for 4 HemesSelect 4HHB – H – hide everythingSelect Heme – Show stickLook His – select and name His F8 and His E7

2

Press S – sequence at bottom screen.Right click – zoom inSelect HEM - hemoglobin

4

Uses molecular modelling

2

Select heme – right click – action – around 5ALook for His F8 and E7 around hemeMake measure for distanceDouble click to display name of atom

Measure bond length/angleMeasure number H2 bonds

Measure bond strengthProtein 1, 2 , 3O structure

Presence of disulfide bondPresence alpha and beta pleated sheet

Protein Data BankProtein database key in - PDB 4HHB

Click here Swiss PDB tutorial

1

2

Uses molecular modelling

1

2

Chemical viewer 3D structure (Swiss PDB)

Download PDB text file

File – open 4HHB pdb downloaded from databank

Window – Control panelRemove – side chainSelect – Group kind – HETATMDisplay – stereo viewShow only selected 4 Heme

Click here down Swiss PDB

Select – Group kind – HistidineSelect – Residue – close to 2ALocate Histidine and make measurement

3

Check for heme and Histidine only from control panel

Select Histidine that are close to ringLocate His F8 and E7 and make measurement

4

Heme

Hemoglobin - 4 chain - 4 heme (porphyrin) - 4 Fe 2+

Fe 2+

Heme (porphyrin)

Hemoglobin - 2 alpha chain - 144 amino acid - 2 beta chain - 146 amino acid

vs

Myoglobin - 1 chain – 1 heme (porphyrin) – 1 Fe2+

- 154 amino acids

HemoglobinMyoglobin

Fe 2+

Heme (porphyrin)

PDB code files

Oxyhemoglobin – 1GZX, 1JY7Deoxyhemoglobin – 1A3N, 4HHB, 2HHB, 1HBB, 1G9V, 101JMyoglobin – 4MBN, 3RGK, 5MBNFetal hemoglobin – 1FDMSickle cell Hemoglobin – 2HBS, 1NEJ

PDB file type for data analysis1

Analyze using Chimera/Pymol/Swiss PDB/Jmol1

Chimera Swiss PDB

JmolPymol

Possible Research Question

Measuring using 3D modelling

Data Collection using 3D modelling

Data Collection using Database

Click here Jmol Click here PyMol

-What is the distance bet Fe and His E7 and F8, and are they the same for diff heme found in hemoglobin/myoglobin-Is His E7/F8 orientation similar for Oxy, Deoxy and Myoglobin.-Is there any differences bet distance/position/orientation of porphyrin ring for Hemoglobin and Myoglobin-How is Fe2+ located, along or out of plane for Hemo/Myoglobin-Is the distance bet Fe and ligand N of porphyrin the same for Hemoglobin/Myoglobin-Is structure/size of porphyrin ring same for α and β chain-Is there any variation in terms of Fe and His E7/F8 for fetal hemoglobin and sickle cell hemoglobin-Why His E7 and F8 are located in such a way across many different species? Are their orientation highly conserved and why?

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Click here Ensembl

Structural similarity and differences bet Hemoglobin and Myoglobin

Myoglobin hemoglobin

Vs

Hemoglobin Chimera Pymol Jmol Swiss PDB Mean

Orientation His/Fe Similar Similar Similar Similar Similar

Bond length N - Fe 2.12A 1.90A 2.02A 2.02A 2.01A

Bond length Fe – E7 5.93A 5.80A 5.45A 5.42A 5.55A

Bond length Fe – F8 2.25A 2.05A 2.10A 2.21A 2.13A

Chimera Swiss PDB

Data source

Myoglobin Chimera Pymol Jmol Swiss PDB Mean

Orientation His/Fe Similar Similar Similar Similar Similar

Bond length N - Fe 2.02A 2.11A 2.15A 2.32 2.14A

Bond length Fe – E7 5.80A 5.71A 5.56A 5.25A 5.25A

Bond length Fe – F8 2.15A 2.25A 2.11A 2.21A 2.21A

His E7

His F8

Fe

N

Possible Research Question Data Collection using 3D modelling

Data Collection using Database

Click here Jmol Click here PyMol

Click here NCBIClick here UCSC

Click here Ensembl

Structural similarity and differences bet Hemoglobin and Myoglobin

Myoglobin hemoglobin

vs

Chimera Swiss PDB

Evaluation and Limitation using 3D modelling

Must use a variety of sources/programme to verify/validate the validity and reliability of data collectedAverage is computed from diff software and checked with database to confirm.Check on methodological limitation using 3D model. (MUST perform 3D Optimization to most stable form structure.Critical and skeptical of result produced by computational chemistry. Major limitation of computation, they assume non-interacting molecule. (Ideal situation, ex molecule in vacuum or isolated state)Most appropriate molecule are those whose coordinates are not theoretical but derive from experimental structural determination(using X ray diffraction)Be careful of predicted arrangement from simulation /3D modelData sources are supported using diff method/3D model/databaseCertain database like NIST and CRC are more reliable source Check if there is a good agreement bet CRC, diff databases and 3D model prediction before making conclusionComputation programme is always based on approximation and we cannot conclusive prove anythingReflect of validity and reliability of dataIs model a true representation of reality?

-What is the distance bet Fe and His E7 and F8, and are they the same for diff heme found in hemoglobin/myoglobin-Is His E7/F8 orientation similar for Oxy, Deoxy and Myoglobin.-Is there any differences bet distance/position/orientation of porphyrin ring for Hemoglobin and Myoglobin-How is Fe2+ located, along or out of plane for Hemo/Myoglobin-Is the distance bet Fe and ligand N of porphyrin the same for Hemoglobin/Myoglobin-Is structure/size of porphyrin ring same for α and β chain-Is there any variation in terms of Fe and His E7/F8 for fetal hemoglobin and sickle cell hemoglobin-Why His E7 and F8 are located in such a way across many different species? Are their orientation highly conserved and why ?

- Porphyrin gp of heterocyclic made of 4 pyrrole subunit- Porphyrin macrocycle has 26 (delocalized) pi electron, obey Hückel rule - It is aromatic, 4n+2 π. (Highly conjugated system)

Heme

PorphyrinHeme = Fe + porphyrins ring

Heme

Heme A Heme B Heme C

Mitochondria- cytochrome c oxidase- electron transport

O2

Heme = Fe + porphyrin ring – carry O2

Fe2+ located

Most abundant Hemoglobin and Myoglobin

Mitochondria- cytochrome c - electron transport

Fetal Hemoglobin (2α22γ2)

Human Hemoglobin (2α2 2β2)

Sickle cell Hemoglobin (2α22βS

2)Myoglobin 1 α chain

Carbaminohemoglobin Carboxyhemoglobin Oxyhemoglobin

Hemoglobin A - 2 alpha and 2 beta chainsHemoglobin A2 - 2 alpha and 2 delta chainsHemoglobin F - 2 alpha and 2 gamma chainsHeme (porphyrin) bind to Fe2+ using 4 nitrogen atom (histidine gp) Porphyrin

- as electron-pair donor - polydentate ligandFe form 2 additional bonds, one on each side of the heme plane. These binding sites call fifth and sixth coordination sites. This hisitidine is referred as proximal Histidine F8The sixth coordination site bind oxygen with His E7 nearby

Deoxygenated hemoglobin formFe2+ - out planeCan’t fit the ring

Heme

Hemoglobin - 4 chain - 4 heme (porphyrin) - 4 Fe 2+

Fe 2+

Heme (porphyrin)

Oxygenated formFe2+ - located in planeFit the ring

Deoxyhemoglobin

Fe2+ out planeCan’t fit the ringOut by 0.06nm

Fe2+ in planeFit the ring

Human hemoglobin - 2 alpha chain - 144 amino acid - 2 beta chain - 146 amino acid

Fe bind to six ligand. 4 with N atom of porphyrinFifth ligand is donated by His 93 (F8) O2 add to Fe as sixth ligandO2 tilt relative to perpendicular of heme plane

His F8His F8 His F8

His E7His E7His E7

Oxyhemoglobin

Heme

Hemoglobin - 4 chain - 4 heme (porphyrin) - 4 Fe 2+

Fe 2+

Heme (porphyrin)

Hemoglobin - 2 alpha chain - 144 amino acid - 2 beta chain - 146 amino acid

Fe bind to six ligand. 4 with N atom of porphyrinFifth ligand is donated by His F8 O2 add to Fe as sixth ligandO2 tilt relative to perpendicular of heme plane

His E7 locate over Fe, force CO to bind to Fe at an angle. This steric hinderance reduce afinity of CO in hemoglobin. O2 bind to Fe at an angle, its binding not affected by presence of His E7.

His (E7)

His (F8)

vs

Myoglobin - 1 chain – 1 heme (porphyrin) - 1 Fe2+

- 154 amino acids

HemoglobinMyoglobin

Fe 2+

Heme (porphyrin)

His (F8)

His (E7)

C≡O