Tópico: Determinación Estructural de Estructura de Proteínas

Principios de la Cristalografía de Proteínas.
E. Rudiño-Piñera rudino@ibt.unam.mx

EXCEPT FOR PROTEIN CRYSTALLOGRAPHY!!!

Yearly growth of Total Structures at the PDB

Yearly growth of Total Structures at the PDB

58747 structures!

Yearly growth of Total Structures Solved by X-ray at the PDB

Yearly growth of Total Structures Solved by X-ray at the PDB

Yearly growth of Total Structures Solved by X-ray at the PDB

85 % from Protein Crystallography!

Protein Crystallography…How a protein structure is generated?

Monocrystals >10 µm sinchrotron

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method. The number of collected frames is related with the crystal symmetry and with the experimental technique used in order to solved…

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases

Electronic density. Model Construction and Refinement . Validation.

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases

Electronic density. Model Construction and Refinement . Validation.

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases

Structural analysis, Biological implications.

Electronic density. Model Construction and Refinement . Validation.

Data Collection (Sinchrotron, in house generator)

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases

Structural analysis, Biological implications.

Electronic density. Model Construction and Refinement . Validation.

Data Collection (Sinchrotron, in house generator)

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases Crucial step.

Structural analysis, Biological implications.

Electronic density. Model Construction and Refinement . Validation.

Data Collection (Sinchrotron, in house generator)

Monocrystals >10 µm sinchrotron

Rotation method.

>80 µm rotating anode

Phases Crucial step.

Structural analysis, Biological implications.

Electronic density. Model Construction and Refinement . Validation.

summary

Phase Problem

summary

Phase Problem

summary

Phase Problem

imaginary axis

Phase Problem: Where the scattering objects are complex (e.g. in molecular crystals) the diffracted radiation suffers a phase shift arising from the spatial distribution of individual scatterers. The amplitudes of the resulting structure factors are directly derivable from the experimental measured intensities of the diffracted beams, but the phases are not.

summary

Phase Problem

Fhkl Φhkl f1 f2

f3

real axis

imaginary axis

Phase Problem: Where the scattering objects are complex (e.g. in molecular crystals) the diffracted radiation suffers a phase shift arising from the spatial distribution of individual scatterers. The amplitudes of the resulting structure factors are directly derivable from the experimental measured intensities of the diffracted beams, but the phases are not.

summary

Phase Problem

Fhkl Φhkl f1 f2

f3

real axis

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

Methods for Phase determination.

– – –

Isomorphous replacement (IR)
Single Isomorphous Replacement (SIR) Multiple Isomorphous Replacement (MIR) Radiation Damage Induced Phasing (RIP)


– –

Anomalous Dispersion (AD)
Single Wave-Length Anomalous Dispersion (SAD) Multiple Wave-Length Anomalous Dispersion (MAD)


– –

IR and AD combination
SIR + SAD = SIRAS (MIRAS) RIP + SAD = RIPAS

• Molecular Replacement. • Direct-Methods (The methods generally exploit constraints or statistical correlations
between the phases of different Fourier components that result from the fact that the scattering density must be a positive real number).

¿Why we used X-rays?

¿Why we used X-rays?

Sinchrotron Radiation

Sinchrotron Radiation:

¡One million times brighter than sun light!

Sinchrotron Radiation:
APS

¡One million times brighter than sun light!

Sinchrotron Radiation:

¡One million times brighter than sun light!

1. Pistola de electrones.

Sinchrotron Radiation:

1

¡One million times brighter than sun light!

1. Pistola de electrones.

Sinchrotron Radiation:

Filamento de tungsteno al vacío.

1

¡One million times brighter than sun light!

1. Pistola de electrones.

Sinchrotron Radiation:

1

¡One million times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC).

Sinchrotron Radiation:

1 2 2 222 2

¡One million times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC).

Sinchrotron Radiation:

1 2 2 222 2

¡One million times brighter than sun light!
Los electrones se aceleran al 99% de la velocidad de la luz con ayuda de microondas

1. Pistola de electrones. 2. Acelerador Lineal (LINAC).

Sinchrotron Radiation:

1 2 2 222 2

¡One million times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster)

Sinchrotron Radiation:
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 1 3 2 2 222 2

¡One million times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster)

Sinchrotron Radiation:
3 3 3 3 3 3
Sincronización de electrones

3

3

3

3 3

3

3 3 3 3 3

3 3

3

3 1 3 2 2 222 2

¡One million times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster)

Sinchrotron Radiation:
3 3 3 3 3 3
Sincronización de electrones

3

3

3

3 3

3

3 3 3 3 3

3 3

3

3 1 3 2 2 222 2

¡One million times brighter than sun light!

Los electrones pasan de 250 MeV hasta 6 GeV con ayuda de microondas.

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster)

Sinchrotron Radiation:
3 3 3 3 3 3
Sincronización de electrones

3

3

3

3 3

3

3 3 3 3 3

3 3

3

3 1 3 2 2 222 2

¡One million times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster) 4. Anillo de almacenamiento.

Sinchrotron Radiation:
4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3
Sincronización de electrones

Bending Magnet

4 3 3

4

3

3

4 4 4 4 3 3 3 3 3 3 3 3

4

4 4 4 4 4 4

3 3

3

3 1 3 2 2 222 2

4 ¡One million 4 times brighter than sun light!

Multipole “Wiggler”

“Undulator”

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster) 4. Anillo de almacenamiento.

Sinchrotron Radiation:
4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3
Sincronización de electrones

4 3 3

4

3

3

4 4 4 4 3 3 3 3 3 3 3 3

4

4 4 4 4 4 4

3 3

3

3 1 3 2 2 222 2

4 ¡One million 4 times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster) 4. Anillo de almacenamiento. 5. Beamline.

Sinchrotron Radiation:
4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3
Sincronización de electrones

4 3 3

4

3

3

4 4 4 4 3 3 3 3 3 3 3 3

4

4 4 4 4 5 4 4 5 5 5

3 3

3

1 3 2 2 222 2

3

4 ¡One million 4 times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster) 4. Anillo de almacenamiento. 5. Beamline. 6. Hutch.

Sinchrotron Radiation:
4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3
Sincronización de electrones

4 3 3

4

3

3

4 4 4 4 3 3 3 3 3 3 3 3

4

4 4 4 4 5 4 4 5 5 5 6

3 3

3

1 3 2 2 222 2

3

4 ¡One million 4 times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster) 4. Anillo de almacenamiento. 5. Beamline. 6. Hutch.

Sinchrotron Radiation:
4 4 4 3 3 3 3 3 4 4 4 4 4 4 4 4 3
Sincronización de electrones

4 3 3

4

3

Hutch

4 4 4

4 4 4 4 3 3 3 3 3 3 3 3

4

4 4 4 4 5 4 4 5 5 5 6

3 3

3

1 3 2 2 222 2

3

4 ¡One million 4 times brighter than sun light!

1. Pistola de electrones. 2. Acelerador Lineal (LINAC). 3. Sincrotrón (Booster) 4. Anillo de almacenamiento. 5. Beamline. 6. Hutch.

Sinchrotron Radiation:
4 4 4 3 3 3 3 3 4 4 4 4 4 4 4 4 3
Sincronización de electrones

4 3 3

4

3

Hutch

4 4 4

4 4 4 4 3 3 3 3 3 3 3 3

4

4 4 4 4 5 4 4 5 5 5 6

3 3

3

1 3 2 2 222 2

3

4 ¡One million 4 times brighter than sun light!

Luz Sincrotrón

The crystal

The crystal

The crystal

The crystal

The crystal
Lisozyme Polarization Izit

300µm

HEWL crystals after 3 hours on a second generation sinchrotron source.

Primary: Secondary:

Haas and Rossmann 1970: lactato deshidrogenasa Acta Cryst B26, 998-1004.

100K nitrogen stream
Dry air or N2

Dry air or N2 Hollow metal pin Metal top hat Pin holding screw Fixing screw Goniometer head

Crystal in fibre loop

X-ray beam

Metal locating pin Flat on side for self-opposing tweezers Lateral vent Magnetic rubber disc

Metal support for magnetic disc

It is CRUCIAL to determined such symmetries BEFORE we attempt to solve phases!!!

BRAGG LAW nλ = 2d sinθ

William Henry Bragg

William Lawrence Bragg

λ

λ

θ d d θ λ = 2d sen θ

θ θ θ θ

Ewald sphere.

1/2

Basic symmetries.

X

X

X X X

X X

Basic symmetries.

Basic symmetries.

X X

Invertion: Imply chirality.

Invertion: Imply chirality.

Reflexion (Mirror):

SYMMETRIES.

Traslation

Rotation-1.

Rotation-2.

Rotation-3.

Rotation-4.

Rotations and axes:
Two-fold rotation axis (360/2=180) Three-fold rotationaxis (360/3=120) Four-fold rotation axis (360/4=90) Six-fold rotation axis (360/6= 60) ¿One-fold rotation axis? ¿five-fold? 360/1=360

m

A group of symmetries could generate “extra” symmetries. m

m

m m

m

1TRN Trypsin

1TRN Trypsin

1TRN Trypsin

1TRN Trypsin

1TRN Trypsin

Space group P4

1TRN Trypsin

Space group P4

Rototranslation:

1A0L β Triptase Space group P41

1A0L β Triptase Space group P41

1A0L β Triptase Space group P41

4/4

1A0L β Triptase Space group P41

3/4

2/4 1/4 0

Screw-rotation axes:

Screw-rotation axes:

Screw-rotation axes:

Screw-rotation axes:

Screw-rotation axes:

7 14 32 Lattices
Unit cell

230 Objects
Symmetry operators

73

65
32 Point groups

7 Crystal systems Alternative lattices objects” (P, I, B, R,etc.) 14 Bravais lattices

“Symmetries for finite

“Infinite spaces”
73 Simorphic groups Translation 230 Space groups

65 Space groups for biomolecules

…after almost 800 experiments performed mainly using a TECAN robot

Our best diffracting crystals:

1.7 Å to 2.3 Å resolution
Owen, R.L. & Garman, E.F. Acta Cryst. (2005). D61, 130-140

crystals grow in: 0.2 M ammonium sulphate, 0.1M Na acetate pH 4.5, 2% MPD, 5% PEG MME 550, 6.0 µl drop size using the hanging-drop method, condition I;

2F13F1

No PENALTY 12 11 10 9 8 7 6 5 4 3 2 1 86 84 10 9 8 7 6 6 5 4 1 0

LATT mC mC tP oC oP mC mP mP mC mP aP aP

a 217.69 37.46 36.98 52.16 36.98 53.11 37.46 36.98 53.11 37.46 36.98 36.98

b 37.46 217.69 37.46 53.11 37.46 52.16 36.98 107.51 52.16 36.98 37.46 37.46

c 36.98 36.98 107.51 107.51 107.51 107.51 107.51 37.46 107.51 107.51 107.51 107.51

alpha 91.0 89.9 90.9 90.8 90.9 90.4 89.7 90.9 89.6 89.7 90.9 89.1

beta gamma 89.9 91.0 89.7 90.4 89.7 90.8 90.9 91.0 90.8 90.9 89.7 89.7 81.0 81.0 91.0 89.3 91.0 89.3 91.0 89.7 90.7 91.0 91.0 89.0

Possible space groups C2 C2 P4,P41,P42,P43,P422, P4212,P4122,P41212,P4222, P42212 P4322,P43212 C222,C2221 P222,P2221,P21212,P212121 C2 P2,P21 P2,P21 C2 P2,P21 P1 P1

Self-rotations calculated in P1

No PENALTY 12 11 10 9 8 7 6 5 4 3 2 1 86 84 10 9 8 7 6 6 5 4 1 0

LATT mC mC tP oC oP mC mP mP mC mP aP aP

a 217.69 37.46 36.98 52.16 36.98 53.11 37.46 36.98 53.11 37.46 36.98 36.98

b 37.46 217.69 37.46 53.11 37.46 52.16 36.98 107.51 52.16 36.98 37.46 37.46

c 36.98 36.98 107.51 107.51 107.51 107.51 107.51 37.46 107.51 107.51 107.51 107.51

alpha 91.0 89.9 90.9 90.8 90.9 90.4 89.7 90.9 89.6 89.7 90.9 89.1

beta gamma 89.9 91.0 89.7 90.4 89.7 90.8 90.9 91.0 90.8 90.9 89.7 89.7 81.0 81.0 91.0 89.3 91.0 89.3 91.0 89.7 90.7 91.0 91.0 89.0

Possible space groups C2 C2 P4,P41,P42,P43,P422, P4212,P4122,P41212,P4222, P42212 P4322,P43212 C222,C2221 P222,P2221,P21212,P212121 C2 P2,P21 P2,P21 C2 P2,P21 P1 P1

P422
Self-rotations calculated in P1

60.000

Native 3 Native 4 Native 5 STATT1-2

45.000

I/sigma(I)

30.000

Axial reflections: h 0 0

15.000

0 0 6 11 h 17 22

40.000

Native 3 Native 4 Native 5 STATT1-2

30.000

I/sigma(I)

Axial reflections: 0 0 l

20.000

10.000

0 0 4 8 12 16 20 24 28 l 32 36 40 44 48 52 56

Helicase (pdb code 1cuk) Space group P4

Helicase (pdb code 1cuk) Space group P4

Helicase (pdb code 1cuk) Artificially moved to P41

60.000

Native 3 Native 4 Native 5 STATT1-2

45.000

I/sigma(I)

30.000

Axial reflections: h 0 0

15.000

0 0 6 11 h 17 22

40.000

Native 3 Native 4 Native 5 STATT1-2

30.000

I/sigma(I)

Axial reflections: 0 0 l

20.000

10.000

0 0 4 8 12 16 20 24 28 l 32 36 40 44 48 52 56

P41212 or P43212 ?

P41212 or P43212 ?

P41212 or P43212 ?

P41212 or P43212 ?

RIP + S-SAD = RIPAD/S (not RAPID!)
Start by assuming: 1. Space group is P41212 or P43212 2. The crystals were not twinned

Best S-SAD solution Best RIP solution Fobs2, FOM2 (statistical error in it, sigma Fobs1, FOM1 (statistical error in it, sigma A weights) A weights) `native’ Origin selection (4 possible for each space group) by phase error test (only one with 69o ; others all 89-90o!)

PHASE COMBINATION
(Sheldrick magic and tangent formula)

PHASE COMBINATION
(Sheldrick magic and tangent formula)

Structure at 1.70 Å

Rigid body minimisation against “before burn” RIP data set at 1.55 Å

Space group Unit cell (Å , o) Solvent content X-ray source Res. range(Å ) Av. multiplicity Compl. (%) I/σ(I) Rmerge (%)

P41212 37.85, 37.85, 108.42, 90, 90, 90 38 % ID14-4 37.74 – 1.55 (1.63 – 1.55) 7.10 (5.50) 98.90 (93.20) 6.00 (1.80) 5.80 (41.60)

…Thinks changes!

…Thinks changes!

2007 1974 …somethings “even” in a constructive way (?) !