Macromolecular Crystallography and Structural Genomics

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Macromolecular Crystallography and Structural
Genomics Recent TrendsProf. D.
VelmuruganDepartment of Crystallography and
BiophysicsUniversity of MadrasGuindy Campus,
Chennai 25.
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• Structural Genomics aims in identifying as many
  new folds as possible.
• This eventually requires faster ways of
  determining the three dimensional structures as
  there are many sequences before us for which
  structural information is not yet available.
• Although Molecular Replacement technique is still
  used in Crystallography for solving homologous
  structures, this method fails if there is not
  sufficient percentage of homology.
• The Multiwavelength Anomalous Diffraction (MAD)
  techniques have taken over the conventional
  Multiple Isomorphous Replacement (MIR) technique.

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• With the advent of high energy synchrotron
  sources and powerful detectors for the diffracted
  intensities, developments in methodologies of
  macromolecular structure determination, there is
  a steep increase in the number of macromolecular
  structures determined and on an average eight
  new structures are deposited in the PDB every day
  and the total entries in the PDB is now around
  29,000.
• Instead of using the three wavelength strategies
  in MAD experiments, the use of single wavelength
  anomalous diffraction using Sulphur anomalous
  scattering is recently proposed. This will reduce
  the data collection time to 1/3rd.
• Also, the judicious use of the radiation damage
  during redundant data measurements in second
  generation synchrotron source and also during
  regular data collection in the third generation
  synchrotron source has been pointed out recently
  (RIP RIPAS).

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Protein Structure Determination

• X-ray crystallography
• NMR spectroscopy
• Neutron diffraction
• Electron microscopy
• Atomic force microscopy

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As the number of available amino acid sequences
exceeds far in number than the number of
available three-dimensional structures,
high-throughput is essential in every aspect of
X-ray crystallography.
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Procedure
Protein Crystal
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The 14 Bravais lattices
2 Monoclinic
1 Triclinic
(Blue numbers correspond o the crystal system)
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The 14 Bravais lattices
3 Orthorhombic
(Blue numbers correspond to the crystal system)
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The 14 Bravais lattices
4 Rhombohedral
5 Tetragonal
6 Hexagonal
(Blue numbers correspond to the crystal system)
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The 14 Bravais lattices
7 Cubic
(Blue numbers correspond to the crystal system)
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Synchrotron radiation
More intense X-rays at shorter wavelengths mean
higher resolution much quicker data collection
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Diffraction Apparatus
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Diffraction Principles
nl 2dsinq
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The diffraction experiment
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The amplitudes of the waves scattered by an atom
to that of an single electron atomic
scattering factor The amplitude of the waves
scattered by all the atoms in a unit cell to that
of a single electron (The vector (amplitude and
phase) representing the overall scattering from a
particular set of Bragg planes) Fhkl
structure factor
The structure factor magnitude F(hk/) is
represented by the length of a vector in the
complex plane.
The phase angle a(hk/) is given by the angle.
measured counterclockwise, between the
positive real axis and the vector F.
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unit cell F (h,k,l) V?x0 ?y0 ?z0
?(x,y,z).exp2?I(hx ky lz).dxdydz A
reflection electron density
V the volume of the unit cell Fhkl the
structure-factor amplitude (proportional to the
square-root of reflection intensities) ahkl the
phase associated with the structure-factor
amplitude FhklWe can measure the amplitudes,
but the phases are lost in the experiment. This
is the phase problem.
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Fourier Transform requires both structure factors
and phases
Electron density calculation
S
S
S
a
?
p
Unknown
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Patterson function

• Patterson space has the same dimension as the
  real-space unit cell
• The peaks in the Patterson map are expressed in
  fraction coordinates
• To avoid confusion, the x, z and z dimensions of
  Patterson vector-space are called (u, v, w).

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What does Patterson function represent?

• It represents a density map of the vectors
  between scattering atoms in the cell
• Patterson density is proportional to the squared
  term of scattering atoms, therefore, the electron
  rich, i.e., heavy atoms, contribute more to the
  patterson map than the light atoms.

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Patterson function no phase info required
Consider phaseless term (h, k, l, F2)
S
S
S
P
No phase term
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Patterson map
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Patterson map symmetry
Patterson map with symmetry
Harker vectors u, v, w 2x, 1/2, 2z
P21 x, y, z -x, y1/2, -z
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Diffracting a Cat
Diffraction data with phase information
Real Diffraction Data
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Reconstructing a Cat
FT
Easy
FT
Hard
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The importance of phases
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Phasing Methodsall assume some prior knowledge
of the electron density or structure
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The Phase Problem

• Diffraction data only records intensity, not
  phase information (half the information is
  missing)
• To reconstruct the image properly you need to
  have the phases (even approx.)
• Guess the phases (molecular replacement)
• Search phase space (direct methods)
• Bootstrap phases (isomorphous replacement)
• Uses differing wavelengths (anomolous disp.)

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Acronyms for phasing techniques

• MR
• SIR
• MIR
• SIRAS
• MIRAS
• MAD
• SAD

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Direct methods

• Based on the positivity and atomicity of electron
  density that leads to phase relationships between
  the (normalized) structure factors (E).
• Used to solve small molecules structures
• Proteins upto 1000 atoms, resolution better than
  1.2 Å
• Used in computer programs (SnB, SHELXD SHARP) to
  find heavy-atom substructure.

Jerome Karle and Herbert A. Hauptman Nobel prize
1985 (chemistry)
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Dm cycle
Density modification procedures (e.g. solvent
flattening and averaging) can be carried out as
part of a cyclic process
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Molecular Replacement (MR)
Used when there is a homology model available
(sequence identity gt 25).

• 1. Orientation of the model in the new unit cell
  (rotation function)
• 2. Translation

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Molecular Replacement (MR)

• MR works because the Fourier transform works in
  both directions.
• Reflections model (density)
• Have to be careful of model bias

New Protein
Coordinates in PDB
MR solution
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Isomorphous replacement

• Why isomorphous replacement, making heavy atom
  derivatives?
• Phase determination
• Calculating FH
• FH FPH-FP
• If HA position is known, FH can be calculated
  from ?(xH, yH, zH) by inverse FT
• HA position determination Patterson function

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HA shifts FP by FH
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Isomorphous Replacement (SIR, MIR)

• Collect data on native crystals (no metals)
• Soak in heavy metal compounds into crystals, go
  to specific sites in the unit cell.
• e.g. Hg, Pt, Au compounds
• The unit cell must remain isomorphous
• Collect data on the derivatives
• As a result, only the intensity of the
  reflections changes but not the indices
• Measure the reflection intensity differences
  between native and derivative data sets.
• Find the position of the heavy atoms in the unit
  cell from the intensity differences.
• generate vector maps (Patterson maps)
• FP HA FP FHA
• Must have at least two heavy atom derivatives
• The main limitations in obtaining accurate
  phasing from MIR is non isomorphism and
  incomplete incorporation (low occupancy) of the
  heavy atom compound.

Native and heavy-atom derivative diffraction
patterns superimposed and shifted
vertically. Note intensity differences for
certain reflections. Note the identical unit
cell (reflection positions). This suggests
isomorphism.
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Isomorphic HA derivatives only changes the
intensity of the diffraction but not the indices
of the reflections
Native crystal HA derivative crystal
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Harker diagram
Once we have an heavy atom structure rH(r), we
can use this to calculate FH(S). In turn, this
allows us to calculate phases for FP and FPH for
each reflection.
Harker construction for SIR
The phase probability distribution shows that SIR
results in a phase ambiguity
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MIR
We can use a second derivative to resolve the
phase ambiguity
Harker construction for multiple isomorphous
replacement (MIR)
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AS Anomalous scattering leads to a breakdown
of Friedels law
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Anomalous scattering data can also be used to
solve the phase ambiguity
Note that the anomalous differences are very
small thus very accurate data are necessary
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?02p
m
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Steps in MAD

• Introduce anomalous scatterer
• Incorporate SeMet in replace of Met
• Incorporate HA eg Hg, Pt, etc
• Take your crystals to a synchrotron beam-line
  (tunable wavelength).
• Collect data sets at 3 separate wavelengths the
  Se (or other HA) absorption peak, edge and
  distant to the peak.
• Measure the differences in Friedel mates to get
  an estimate of the phases for the Se atoms.
• These differences are quite small so one need to
  collect a lot of data (completeness, redundancy)
  to get a good estimate of the error associated
  with each measurement.
• Use the Se positions to obtain phase estimates
  for the protein atoms.

Atomic scattering factor 3 terms
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Advantages of MAD

• All data is collected from one crystal
• Perfect isomorphism
• Fast
• Easily interpretable electron density maps
  obtained right away.

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SADSingle-wavelength anomalous diffraction
(SAD) phasing has become increasingly popular in
protein crystallography.Two main steps 1)
obtaining the initial phases 2) improving the
electron density map calculated with
initial phases.

• The essential point is to break the intrinsic
  phase ambiguity.
• Two kinds of phase information enables the
  discrimination of phase doublets from SAD data
  prior to density modification.
• From heavy atoms (expressed by Sim distribution)
• From direct methods phase relationships
  (expressed by Cochran distribution)

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Breaking the OAS phase ambiguity
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OASIS (CCP4 Supported Program) DESCRIPTION
OASIS is a computer program for breaking phase
ambiguity in One-wavelength Anomalous Scattering
or Single Isomorphous Replacement (Substitution)
protein data. The phase problem is reduced to a
sign problem once the anomalous-scatterer or the
replacing-heavy-atom sites are located. OASIS
applies a direct method procedure to break the
phase ambiguity intrinsic to OAS or SIR data.
REFERENCES Fan, H. F. and Gu, Y. X. (1985)
Combining direct methods with isomorphous
replacement or anomalous scattering data III.
The incorporation of partial structure
information, Acta Cryst. A41, 280-284. Fan H.
F., Hao, Q., Gu, Y. X., Qian, J. Z., Zheng, C. D.
and Ke, H. (1990) Combining direct methods with
isomorphous replacement or anomalous scattering
data VII. Ab initio phasing of the OAS data from
a small protein, Acta Cryst. A46, 935-939. Y. -D.
Liu, I. Harvey, Y. -X. Gu, C. -D. Zheng, Y. -Z.
He, H. -F. Fan, S. S. Hasnain and Q. Hao (1999)
Is single-wavelength anomalous scattering
sufficient for solving phases? A comparison of
different methods for a 2.1 A structure solution,
Acta Cryst. D55, 1620- 1622. AUTHORS Q. Hao (1,
2), Y. X. Gu, C. D. Zheng H. F. Fan (2)
(1) School of Applied Sciences, De Montfort
University, Leicester LE1 9BH, England.
(2) Institute of Physics, Chinese Academy of
Sciences, Beijing 100080, P. R. China. Email
qhao_at_dmu.ac.uk or fan_at_aphy.iphy.ac.cn
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The first example of solving an unknown protein
by direct-method phasing of the 2.1Å OAS data
Rusticyanin, MW 16.8 kDa SG P21
a32.43, b60.68, c38.01Å b107.82o
Anomalous scatterer Cu
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Comparison of OAS and MAD phasing
(data from Dr. S. Ealick)
MAD phasing Direct-method
OAS phasing
Ompdc
Pure
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Radiation damage Induced Phasing (RIP)

• Radiation damage has been a curse of
  macromolecular crystallography from its early
  days.
• The X-ray radiation damage of cystals can be
  caused by he breakage of covalent bonds as an
  immediate consequence of the absorption of an
  X-ray quantum (a primary effect) of by the
  destructive effect of the propogation of radicals
  throughout the crystal (a secondary effect).
• Total dose and dose rate play a role in the
  amount of radiation damage inflicted on a protein
  crystal.

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• The most pronounced structural changes observed
  were disulphide-bond breakage and associated
  main-chain and side-chain movements as well as
  decarboxylation of aspartate and glutamate
  residues.
• The structural changes induced on the sulphur
  atoms were successfully used to obtain
  high-quality phase estimates through an RIP
  (Radiation damage Induced Phasing) procedure.

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Radiation damage Induced Phasing with Anomalous
Scattering (RIPAS)

• Substructure solution and phasing procedure using
  a combination of anomalous scattering and
  radiation damage induced isomorphous differences.
• RIPAS strategy is beneficial for both locating
  the substructure and subsequent phasing.

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Experimental electron density before
solvent flattering with SAD (left), RIP (middle)
and RIPAS (right) phases for the (a) CS
(thaumatin crystal soaked in a diluted
N-iodisuccinamide solution) thaumatin data
(b) IC thaumatin (iodinated crystallized
thaumatin)
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Methods of phase improvement
It is not always (!) possible to recognise
features in a first electron density map. There
are however ways of improving the map (phases)

• Solvent Flattening
• Histogram matching
• Non-crystallographic symmetry (NCS) Averaging
• these methods can result in dramatic
  improvements in the clarity of the electron
  density map.

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1. Solvent flattening. Protein crystals contain
large amounts of solvent this will in general be
disordered, and so will not contribute to the
crystal diffraction. By knowing the protein
content of the crystal, it is therefore possible
to determine the threshold density below which is
noise points with density below the threshold
are set to a suitable average value. This is
particularly useful for locating molecular
boundaries.
2. Averaging. If the asymmetric unit possesses
more than one molecule, the equivalencing of the
various copies can lead to dramatic improvement
in the map and the phases.
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Improvement in electron density after solvent
flattening and histogram matching
Before
Green solvent envelope
After
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Interpretation of the Electron Density(Building
the Model)

• Lots of fun!
• Trace the main-chain
• Try to recognize the amino acid sequence in the
  density.
• Programs- Xtal view, O

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The effect of resolution of the quality of the
electron density map
2.0 Å
1.5 Å
1.2 Å
5.0 Å see shape of molecule 3.0 Å see
main-chain and some side chains 2.5 Å see
main-chain carbonyls 1.5 Å atomic resolution.
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Resolution
1.2 Å
2 Å
3 Å
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Atomic resolution
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Fitting side chains, adding waters

• If the density is good enough you can recognize
  alternate conformations for side-chains.
• Hydrogens are not seen in the density, except in
  ultra-high resolutions structures lt 1.0 Å.
• Ordered Waters are seen on the surface and
  occasionally in the interior of the protein.
• At 2.0 Å resolution or better 1 water /
  residue.
• Waters molecules play a big role in protein
  stability and enzyme catalysis.
• Because the density depends on experimental
  phases which has error associated with them.
• The first model can have many errors.
• Therefore it is essential to refine the atomic
  positions and their thermal parameters.

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Chain Tracing
Electron Chain Final Density Trace
Model
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Maps coefficients used to minimize model bias
2Fo Fc most common map seen in paper. Fo
Fc (difference map) used with the above map
to detect errors
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Refinement Cycle
Refinement Improving the agreement between the
model and the experimental density. Compare Fobs
(From reflection Intensities) to Fcalc
(Calculated from the model) Least squares
minimization Simulated Annealing / Molecular
dynamics Rfactor numerical indicator to follow
progress of refinement agreement between data
and model
data
model
data
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Refinement
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Refinement
iterations
R
R S(Fo-Fc)/S(Fo)
Fc calculated structure factor
Fo observed structure factor
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Protein Data Base growth
Molecular Biology cloning of genes / over
expression of proteins Synchrotron Radiation
MAD phasing, smaller crystals Cryo-cooling of
crystals collect data from 1 crystal,
increase order. Instrumentational and
software improvements Increase in the number of
labs using the technique
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• Due to the advent of synchrotron radiation and
  due to the seleno-methionine derivatization
  technique, the total number of protein structures
  deposited in the PDB from 1980 onwards has
  increased catastrophically.
• MAD technique played a major role in this. At
  present nearly 100 new structures are deposited
  every week.

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• THANK YOU