# macromolecular crystallography and structural genomics – recent trends prof. d. velmurugan...

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Macromolecular Crystallography and Structural Genomics Recent Trends

Prof. D. Velmurugan

Department of Crystallography and BiophysicsUniversity of MadrasGuindy Campus, Chennai 25.

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.

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).

Protein Structure DeterminationX-ray crystallographyNMR spectroscopyNeutron diffractionElectron microscopyAtomic force microscopy

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.

ProcedureProtein Crystal

The 14 Bravais lattices

1: Triclinic2: Monoclinic(Blue numbers correspond o the crystal system)

The 14 Bravais lattices

3: Orthorhombic(Blue numbers correspond to the crystal system)

The 14 Bravais lattices

4: Rhombohedral5: Tetragonal6: Hexagonal(Blue numbers correspond to the crystal system)

The 14 Bravais lattices

7: Cubic(Blue numbers correspond to the crystal system)

Synchrotron radiationMore intense X-rays at shorter wavelengths mean higher resolution & much quicker data collection

Diffraction Apparatus

Diffraction Principlesnl = 2dsinq

The diffraction experiment

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.The amplitudes of the waves scattered by an atom to that of an single electron atomic scattering factorThe 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

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 |Fhkl|We can measure the amplitudes, but the phases are lost in the experiment. This is the phase problem. unit cellF (h,k,l) = Vx=0 y=0 z=0 (x,y,z).exp[2I(hx + ky + lz)].dxdydz A reflection electron density

Fourier Transform requires both structure factors and phasesUnknownElectron density calculation

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

What does Patterson function represent?It represents a density map of the vectors between scattering atoms in the cellPatterson 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.

Patterson function no phase info requiredPConsider phaseless term (h, k, l, F2)No phase term

Patterson map

Patterson map symmetryP21

x, y, z-x, y+1/2, -zPatterson map with symmetryHarker vectors

u, v, w2x, 1/2, 2z

Diffracting a CatDiffractiondata withphase informationReal DiffractionData

Reconstructing a CatFTFTEasyHard

The importance of phases

Phasing Methodsall assume some prior knowledge of the electron density or structure

The Phase ProblemDiffraction 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.)

Acronyms for phasing techniques MRSIRMIRSIRASMIRASMADSAD

Direct methodsBased 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

Used in computer programs (SnB, SHELXD SHARP) to find heavy-atom substructure.

Jerome Karle and Herbert A. HauptmanNobel prize 1985 (chemistry)

Dm cycleDensity modification procedures (e.g. solvent flattening and averaging) can be carried out as part of a cyclic process

Molecular Replacement (MR)1. Orientation of the model in the new unit cell (rotation function)2. TranslationUsed when there is a homology model available (sequence identity > 25%).

Molecular Replacement (MR)MR works because the Fourier transform works in both directions. Reflections model (density)

Have to be careful of model biasNew ProteinCoordinates in PDBMR solution

Isomorphous replacementWhy isomorphous replacement, making heavy atom derivatives?Phase determinationCalculating FHFH= FPH-FP If HA position is known, FH can be calculated from (xH, yH, zH) by inverse FTHA position determination Patterson function

HA shifts FP by FH

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 isomorphousCollect data on the derivativesAs a result, only the intensity of the reflections changes but not the indicesMeasure 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 derivativesThe 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 derivativediffraction patterns superimposed and shifted vertically.Note: intensity differences for certain reflections.Note: the identical unit cell (reflection positions). This suggests isomorphism.

Isomorphic HA derivatives only changes the intensity of the diffraction but not the indices of the reflectionsNative crystalHA derivative crystal

Harker diagramOnce 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 SIRThe phase probability distribution shows that SIR results in a phase ambiguity

MIRWe can use a second derivative to resolve the phase ambiguityHarker construction formultiple isomorphous replacement (MIR)

ASAnomalous scattering leads to a breakdown of Friedels law

Anomalous scattering data can also be used to solve the phase ambiguityNote that the anomalous differences are very small; thus very accurate data are necessary

02pm=

Steps in MADIntroduce anomalous scattererIncorporate SeMet in replace of MetIncorporate 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

Advantages of MADAll data is collected from one crystalPerfect isomorphismFastEasily interpretable electron density maps obtained right away.

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 modific

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