X-ray crystallography

What are the four levels of proteins structure?

primary, secondary, tertiary, quaternary

What is primary structure?

sequence of amino acids

What is the secondary structure?

local folding arrangement, relationship of an amino acid to its neighbours in the chain (alpha helix, beta sheet, beta loop)

What is tertiary structure?

complete 3D structure of protein chain (full polypeptide)

What is quaternary structure

spatial arrangement of multiple chains found in some proteins

What can structure tell us?

•Understand biological processes at the basic level

•Understand disease at an atomic level

•Help develop new drugs

•Engineer new and improved proteins for various applications

What does atomic resolution structural biology do?

determine atomic structure to analyse why molecules interact

What is X-ray crystallography

technique used to determine the 3D structure of a molecule
to do it, purified samples of DNA are crystallized and bombarded X-rays

How do we get structure from X-ray crystallography?

Electrons from atoms scatter the X-rays, so we see the electron cloud around the molecule -we build a model of atom positions to interpret the image.

What are the aims of XRC?

to be able to use protein structures from database in a knowledge manner, to make it easier to understand protein structure and limitation of data

Bernal and Hodgkin report the first X-ray diffraction when and of what?

1934 and from a protein (pepsin), the birth of protein crystallography.

Perutz shows the use of heavy atoms when and for what?

1953 and be used to solve the structures of proteins.

What distances do crystallographers use and what are the wavelengths?

angstrom (0.5-1.5 Angstrom)

What wavelengths are x-rays?

0.1 Angstrom ->100angstrom

How do you convert from angstrom to m?

1x10^-10m

How do microscopes work?

•Light strikes the object and is diffracted in various directions

•The lens collects the diffracted rays and reassembles them to form an image

Why can't we do x-ray microscopy?

no x-ray lens so can detect diffraction but need different way to assemble image

Why do we need crystal in x-ray?

to amplify signal

How does x-ray crystallography work?

•High-powered X-rays are aimed at a tiny crystal containing trillions of identical molecules

•Crystal scatters the X-rays onto an electronic detector

•Electronic detector

What is a good protein sample?

pure, defined buffer, defined concentration, no aggregation, need lots of pure protein

How do you improve having no aggregation aka dynamic light scattering, size exclusion

salt, pH, temperature, detergent, batch, cofactors, binding partners, mutagenesis

How do you check how pure your protein is?

•SDS-PAGE, Mono Q, Iso Electric Focusing, mass spectroscopy

How much protein do you need on average and for how many conditions?

1-3mg for 2000 conditions

What are the steps in solving protein structure in x-ray crystallography?

purify protein, grow a crystal, collect and process diffraction data, phase the diffraction data, calculate an electron density map, build and refine the structure

How does crystal act as amplifier?

•A crystal arranges huge numbers of molecules in the same orientation, so that scattered waves can add up in phase and raise the signal to a measurable level

What is a crystal?

a solid material whose constituents (atoms, molecules, or ions) are arranged in a highly ordered lattice that extends in all directions.

What are the types of symmetry in packing of molecules in the crystal?

mirror, rotation, reflection and inversion

What are proteins (two properties regarding structure)?

3d and chiral

What is a crystal unit cell defined by?

•its cell constants and is the building block for the whole crystal

•Edges: a, b, c

Angles: a, b,g

What generates space group symmetry?

Combining point group and Bravais lattice symmetries generates space group symmetry

What is the space group a description of

complete symmetry of ideal crystal

How many space groups are there and how many are possible for chiral molecules

230 different space groups (first derived in nineteenth century), of which only 65 are possible for chiral molecules

What is the resulting diffracted wave the sum of?

sum of diffraction from interaction with matter all the way along the plane

What are some caveats of x-ray crystallography?

•Sometimes a protein will adopt a different structure in a crystal than it does in its natural environment

•Crystallography gives you a static snapshot of a protein's structure

How are unit cells defined?

in terms of lengths along x,y and z crystal axes and angles between them

What are the crystallographic symmetry symbols as viewed downed symmetry axis and which are rotation only and which are rotation and translation?

rotation only:2, 3, 4 and 6

rotation and translation: 2_1, 3_1, 3_2, 4_1, 4_2, 4_3, 6_1, 6_2, 6_3, 6_4, 6_5

What are the symmetry symbols as viewed perpendicular to symmetry axis?

2_1, 4_1

How many crystal systems are there?

7

What are the seven crystal systems?

triclinic, monoclinic, orthorhombic, tetragonal, hexagonal, trigonal, cubic

How many Bravais lattices are there?

14

How come there are 14 Bravais lattices but 7 crystal systems?

due to centring, so monoclinic has p and c, orthorhombic has p, l, c, F and tetragonal has p and l, and cubic has p, l, F

how do you interpret triclinic space group notation?

Following the lattice type, the remaining symbol is either 1 or -1 showing the absence or presence of an inversion centre, respectively

How do you interpret monoclinic space group notation?

After the lattice type, the remaining part of the space group symbol indicates symmetry with respect to the unique axis direction (2, m or 2/m)

How do you interpret orthorhombic space group notation?

After the lattice type, there are three parts to the space group symbol indicating the symmetry with respect to the x, y, and z axis directions, respectively. (222, mm2(or m2m or 2mm), mmm)

How do you interpret tetragonal space group notation?

subdivided into two groups: Those with and without additional symmetry with respect to the x and y axes. The fourfold symmetry is always chosen to lie parallel to the z axis and is specified second in the space group symbol after the lattice type. For those space groups with symmetry along other the other axes, the next part of the symbol indicates the symmetry with respect to both the x and y axes. The remaining part of the symbol indicates the symmetry with respect to both of the diagonals between the x and y axes.
(P4, P-4, P4/m, P422, P4mm, P-42m (or P-4m2), P4/mmm.)

How do you interpret trigonal + Rhombohedral space group notation?

The threefold symmetry is always chosen to lie parallel to the z axis and the symbol for it follows the lattice type in the space group symbol. Additional symmetry elements may be lie parallel to the x and y axes or perpendicular to them. The order of the last two components of the space group symbol is used to distinguish the two possibilities
(P3, P-3, P321, P312, P3m1, P31m, P-3m1, P-31m.)

How do you interpret hexagonal space group notation?

similar to that of the trigonal space group symbols except that the symmetry with respect to the z axis is now of order six.
(P6, P-6, P6/m, P622, P6mm, P-62m (or P-6m2), P6/mmm.)

How do you interpret cubic space group notation?

lattice type (P, F, or I) followed by symmetry with respect to the x, y, and z axes, then the threefold symmetry of the body diagonals, followed lastly by any symmetry with respect to the face diagonals if present
(P23, Pm-3, P432, P-43m, Pm3m.)

What combines through space-group specific symmetry to generate unit cell?

ASU (asymmetric unit)

In protein crystallography what do we determine the structure of and why can this be a problem?

asymmetric unit, and it may not correspond to biologically relevant assembly

Space group P1 has no rotational symmetry what does this mean for ASU?

ASU is equivalent to unit cell

how do you calculate matthew coefficient?

unit cell volume(angstrom cubed)/(molecular weight(daltons) x no ASU(defined by space group) x no. moles per ASU)

How do low solvent content crystals and high solvent content crystals differ?

•Low solvent content crystals tend to be highly ordered and diffract well
•High solvent content crystals tend to be less ordered - fewer crystal contacts - leading to weaker diffraction

What are crystallising agents?

salts, long chain organic polymers, organic solvents

How does high salt precipitate a protein?

The salt ions order water molecules around them, leaving less unstructured water to solubilize the protein

How does organic solvents precipitate a protein?

•These effectively dilute water with a less polar, less H- bond capable solvent with lower dielectric etc.

How does long chain organic polymers precipitate a protein?

•PEG prefers to writhe over a large volume of space

•Taking the protein out of solution frees up more space for PEG and is energetically favoured

What other factors affect crystallisation?

protein concentration, pH, temperature, presence of ligands

How does protein concentration affect crystallisation?

Need less precipitant to precipitate the more concentrated the protein

How does pH affect crystallisation?

Changing the pH adds/removes protons from individual residues, possibly creating new salt bridges/H-bonds

How does temperature affect crystallisation?

As temperature changes, so do the enthalpic and entropic contributions to Delta G of crystallization

How does the presence of ligands affect crystallisation?

•Ligands may lock the protein into one conformation, which can help crystallization

What is screening

Start with commercial screening kits derived from extensive practical experience; there are hundreds mixtures covering wide range of conditions

How does vapour diffusion work?

A drop composed of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent.

How does the loss of water in vapour diffusion help with crystallisation?

the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir

What is optimisation

Once a lead condition is found form screening process expansion (pH and concentration of precipitant etc.) will be carried out

When screening what do you look for?

crystal leads, anything that appears crystalline

Do all proteins crystalline?

no

How do you improve size and diffraction

•Systematic variation of all concentrations and pH

•Additive screens and detergent screens

•Temperature

•Seeding with crushed crystals (micro seeding)

•Dialysis, batch, sitting drop

What do you do if protein doesn't crystallise?

•Check purity and stability

•Remove cysteins and other trouble makers

•Remove flexible parts

•Try single domains

•Try physiologically relevant complexes

Why are protein crystals very fragile and what are they grown from?

since they are half water, and grown from super saturated conditions

What is benefit of using robotic crystallisation?

good for screening since can go through huge number of conditions in single plate since so many wells

Obtaining diffraction quality crystals is normally what?(disadvantage)

rate limiting(can take months)

what is x-ray crystallography used for?

to strudy biomolecular structures using crystals

Why do we use x-rays?

have wavelength similar to inter-atomic distances

How is x-rays emitted onto crystal in x-ray crystallography?

coherent monochromatic beam of x-rays (focused beam)

how do you obtain diffraction data from crystals?

using miller indices due to assembly of a set of planes defined by miller indices

What is Bragg's law?

•Planes in a crystal are separated by distance (d)

•Incident beam meets the plane at angle q

•When light (in our case X-rays) is reflected from a mirror, the angle of incidence is equal to the angle of reflection

Why are diffraction spots called reflections?

since crystal is composed of lots of 'mirrors' that reflect x-rays

When would wave 1 and 2 result in constructive diffraction?

if they add to make lambda, since they are in phase

For any angle of incidence of the beam, only a subset of planes meet the Bragg law conditions, what does this mean for our crystals

crystals must be rotated to vary the angle of incidence of the beam

what does d stand for ?

resolution measured in angstrom

How do you know what d is?

dictated by crystal

What is capturing diffractions influenced by?

•Distance between crystal and detector

•Size of the detector

When do we see 'in-phase' diffraction from sets of planes?

at the angle of reflection and we only see that reflection/spot if the detector is in the right place to catch it

What is the size of spot directly related to?

amount of matter on that set of planes

How do we used x-ray crystallography for proteins?

by rotating it in order to collect large number of data to represent structure properly

How is laboratory source used in x-ray crystallography?

rotating anode -Electron beam hitting copper (Cu) target

Fixed wavelength1.5418Å

How is synchrotron used in x-ray crystallography?

-Circular accelerator + Undulator

-Much more intense

-Narrow beam

-Wavelength tunable

How is free electron laser used in x-ray crytallography?

-Linear accelerator + Undulator

-Even more intense

-Femtosecond pulses

What are the types of detectors to produce digital images?

Image plate (phosphorimager)

» CCD (photon coupled)

» Solid state (direct)

What is high energy synchrotron radiation (SR)-x-rays

Electromagnetic radiation when charged particles (electrons) are radially accelerated (moved in a circular path under vacuum) - high brilliance

What happens in diffraction of x-rays?

x-rays deflected by electrons as they pass through protein crystal, and then absorbed as pattern of dots (diffraction pattern) by a photon detector

What are the features you look for in diffraction images?

discrete spots, intensities are different from spot to spot (general: decrease from centre to edge)

What is the letter for Low and High resolution?

low=L, high=R

Where is the high resolution?

round edge since more spots

What are the position and intensity of spots related to?

electrons of each individual protein atom

What happens in data collection in x-ray diffraction?

many frames at different crystal angles

If a protein is diffracted from all angles, analysis of the diffraction patterns will provide what?

a template of the electron densities within the protein

Spacing between spots contain information about what?

geometry of crystal