Module 4- Structural Biology And Biomedical Research

Question 1

Methods of determining protein structure

Answer 1

X-ray crystallography
Cryo-electron microscopy
Nuclear magnetic resonance

Question 2

How does X-ray crystallography work

Answer 2

Diffraction occurs from protein crystal defracting light
Diffraction pattern is recorded
Diffraction compared with optical microscopy
Crystal is made by being frozen

Question 3

Crystal diffraction pattern

Answer 3

Spots vary in intensity, size, location and scattering angle
Can predict where spots will be in a pattern
To measure the diffraction, measure the intensity and location of spots
Diffracted spots transformed into electron density map with which a model is built from

Question 4

Superimposed meaning

Answer 4

Put on top of one another

Question 5

superposed meaning

Answer 5

Align each and put on top of one another. Can get RMSD when done. RMSD increases with more difference in structure

Question 6

Advantages of protein crystallography

Answer 6

Can obtain high resolution structures
High structural detail possible
Good for studies on bound ligands
Often produced nobel prize research

Question 7

Disadvantages of protein crystallography

Answer 7

Need pure stable protein
Need protein crystals
Need diffraction equipment
For membrane proteins- very hard to obtain crystals

Question 8

Basis of cryo electron microscopy

Answer 8

Single particle images generated by TEM
Alignment of single particles and 2D class average to get 3D
3D classification average to get 3D refinement and final high-resolution structure (20S proteasome)
Can get atomic structures from this

Question 9

Pros of cryo electron microscopy

Answer 9

Crystals not required
Yields atomic model
Good for membrane proteins

Question 10

Cons of cryo electron microscopy

Answer 10

Resolution limited
Size limited
Expensive
Sample prep is hard

Question 11

Pros of NMR

Answer 11

No crystal needed
No cryoEM required

Question 12

Cons of NMR

Answer 12

Expensive
Isotopic labelling often needed
Structure not as accurately determined generally as X-ray

Question 13

Goal of structural study

Answer 13

Explain a biological or biochemical question
Framework to understand important scientific question

Question 14

Steps in x-ray crystallography structural determination

Answer 14

Purification of protein
Crystallization
Measuring diffraction
Phasing- electron density map
Building an atomic model
Refining the model
Interpreting the structure

Question 15

Protein purification in crystallization

Answer 15

Done many ways; over-expression in cells, affinity tags, performance hardware
Want a soluble, unaggregated uniform pure protein
5-10mg at ~95% purity, ~10mg/mL
Stable in aqueous buffer

Question 16

How is protein crystallization carried out

Answer 16

Also known as controlled precipitation hoping crystal formation is energetically favourable over amorphous precipitate
Equilibration with conc salt, PEG in small wells (10ug) in plates
Trial and error
Crystallization is rate limiting step

Question 17

Common precipitants in protein crystallization

Answer 17

PEG (polyethylene glycol polymer) 400, 8000, 20000
Ammonium sulfate
PEG/ lithium chloride
Phosphate
Organic solvents

Question 18

Variables in protein crystallization

Answer 18

Protein concentration
Precipitant concentration
pH
Ligands, additives
Temperature

Question 19

Saturation in crystallization (saturated, unsaturated, supersaturated)

Answer 19

Saturated for a substance= can no longer dissolve any more of it
Unsaturated= can dissolve more
Supersaturated= has somehow dissolved too much, not stable situation
Crysallization is move from supersaturated state to saturated state- at end have crystals more ordered than the precipitant and saturated protein

Question 20

Measuring diffraction/ data collection in crystallography (diffraction collection)

Answer 20

Crystals mounted in path of x-ray beam
Rotated to yield diffracted spots of reflections
Crystals must diffract well and be stable in x-ray
Often flash frozen to make stable
Spots away from beam stop in diffraction beam= high resolution. Close= low resolution

Question 21

Law associated with diffraction and what it means

Answer 21

Braggs law n(wavelength)=2d sin x angle
When they are equal= constructive interference
Proteins are packaged in a lattice separated by d in angstroms
Only some angles give spots

Question 22

Lattice

Answer 22

Packaging arrangement in a crystal
When crystallizing occurs protein molecules pack into lattice based on charge and shape
Packing extends along 3 axes (a b c)

Question 23

Unit cell

Answer 23

Unique unit of volume or area which can be used to build up a complete lattice
Built up by translation along a b c axes
3D analogy: bricks in brick wall
2D analogy: tiles on floor
Protein crystal composed of unit cell of protein molecules stacked together

Question 24

Asymmetric unit

Answer 24

Smallest part of unit cell that can be used to create the complete unit cell when all the internal symmetry elements in the crystal are applied

Question 25

Space group

Answer 25

Combination of unit cell symmetry and crystal structure lattice that describes a given crystal under study is called space group

Question 26

Phasing and intensity in crystallography- what it is

Answer 26

X-rays are magnetic wave with intensity and phase Phase is where wave peaks are relative to each other. If they align, get bigger wave, if not then cancel each other out and get nothing Need intensity and wave of diffracted x-ray for electron density- structure factor Diffraction has intensity info

Question 27

Structure factor in crystallography

Answer 27

F or IFI Numerical value of structure factor proportional to square root of intensity of diffracted wave Phaase angle is 0-360 If F measured experimentally= Fo (observed) If F calculated from model= Fc (calculated) F can be added together to calculate electron density maps then used to create protein models

Question 28

Electron density in crystallography

Answer 28

Electron cloud that surrounds each atom Molecules modelled into density like hands in gloves by computer programmes Protein structures are atomic models that have been built in an electron density map

Question 29

How is phasing of diffraction pattern done

Answer 29

Phase of reflections lost in data collection, getting it back is phasing Good phasing= good maps= accurate structures Patterson or direct methods, MIR, MAD or MR

Question 30

What is patterson or direct methods for phasing diffraction pattern

Answer 30

Math function solved to locate atoms in unit cell Once located, phases calculated Works with small number of atoms- not proteins directly Useful to find heavy atoms within protein eg metals

Question 31

What is multiple isomorphous replacement MIR for phasing

Answer 31

Based on soaking heavy atoms into protein crystals Requires multiple crystals and data sets Allows patterson method to be used Uses data from 2 kinds of crystals, when combined, data solved by patterson to locate heavy atoms Heavy atom locations then lead to locating protein atoms

Question 32

What is multiwavelength anomalous dispersion MAD for phasing

Answer 32

Based on use of selenomethionine in place of methionine Related to MIR- doesnt require soaking crystal in heavy atom solution Requires collection at synchrotron Collect 4 datasets at wavelengths to maximise effects of selenium MAD variant SAD uses 1 wavelength at peak MAD/SAD grown to dominate phasing of structures

Question 33

What is molecular replacement MR for phasing

Answer 33

Uses already solved structures- source for starting phases Requires structural homology Good for mutants of known structures Good for high identity between sequences If initial model is wrong leads to structures with errors= phase bias Most common method to solve homologous structures

Question 34

What is amplitude

Answer 34

Amplitude is the magnitude of structure factor

Question 35

Fitting crystallography to an electron density map (building the model)

Answer 35

Phasing yields empty density hopefully following chain of structure of protein Building means inserting atomic structure into envelope of electron density Room for error, manual checking required Electron density map from combining different amplitudes and phases

Question 36

Types of electron density maps- combining different amplitudes and phases

Answer 36

Fo Fo-Fc= difference map 2Fo-Fc Omit map 2Fo-Fc with omit phases

Question 37

What is an Fo map

Answer 37

measured intensities/ observed amplitudes with phases from model usually combined with a(calc)- phase from MIR/MR/Paterson

Question 38

What is an Fo-Fc map/ difference map

Answer 38

phases from model and observed-calculated amplitude, will see +ve and -ve density Positive means may need to add atoms (green) Negative means may need to remove atoms (red) (Fo-Fc)acalc

Question 39

What is a 2Fo-Fc map

Answer 39

Observed- calculated amplitude, phases from model Has features of Fo and difference map Most common map in protein work (2Fo-Fc)acalc

Question 40

What is an omit map

Answer 40

Special map used to test part of the model that seems uncertain Intensities are 2Fo-Fc and phases from a model with some atoms left out Things left out remain in the map if supposed to be in structure (2Fo-Fc)acalc [part of model omitted]

Question 41

What is refinement

Answer 41

Moving atoms to minimise difference between theoretical diffraction data calculated from observed model and data measured or observed experimentally Done after initial fitting to density Minimise (Fo-Fc): perfect model Fo=Fc

Question 42

More on how refinement is done

Answer 42

Shifting protein and solvent atoms into best xyz positions for atoms while preserving correct geometry Also need to calculate occupancy (what % of unit cells have atom/ligand etc) and temperature factor (relates to thermal motion of atoms) for each atom

Question 43

More on temperature factors

Answer 43

B-factors Number assigned to each atom in a structure, measuring thermal motion 0-100 Atoms with high motion show different in maps Can indicate disorder or bad data B<20 excellent, <20-40 common, >60 mobile or disordered Waters have high B factors- can be good Plot of B vs residue number tells mobile regions in protein structure

Question 44

Reporting crystallography results

Answer 44

Result of refinement is optimised model Recorded in PDB file format Chain no, residue, atom, xyz, occupancy, B-factor Deposited in PDB along with structure factors

Question 45

Crystallography interpretation and analysis

Answer 45

After completion of refinment analyse structure quality, topology, tertiary structure and active site Develop a model Devise experiments to test model

Question 46

Three ways to determine crystallography structure quality

Answer 46

Quality of X-ray diffraction data sets Quality of X-ray structure determination and refinment Quality of overall protein model

Question 47

Ways that quality of data sets is measured (how well are spots measured)

Answer 47

Resolution I/sigma (signal to noise) Completeness Multiplicity R(merge) R(pim) CC(1/2)

Question 48

Resolution

Answer 48

Indicator of quality High resolution- away from beam stop and low number= fine structural details Low resolution- close to beam stop and high number= coarse structural details Determined by Bragg angle of diffracted sports Higher angle> higher resolution

Question 49

What different angstrom measurements correlate to in resolution

Answer 49

6= overall shape 3= trace chain 2.5= carbonyl 2.0= holes in aromatics 1.5= see individual atoms 1= see H atoms

Question 50

Signal to noise

Answer 50

I= intensity, sigma= standard deviation of I Ratio of signal to noise Can be per reflection, per data shell, per data set Common high resolution cut off is 2-3 Full data sets: 5-10 is fair, >20 is good Analysed by computer programmes

Question 51

Completeness and redundancy

Answer 51

How much of available data is being measured How many times is each reflection being measured Consider in context of signal to noise Analysed by computer Gives % of completeness shell Average multitude= redundancy

Question 52

R-merge

Answer 52

How well spots which are measured multiple times agree % of difference in intensities Historically important, goal was 10% or less Big data sets have high numbers- newer stats R-pim and CC developed for big data sets so this one no longer as important

Question 53

CC(1/2) and R-pim

Answer 53

R-pim is redundancy corrected R-merge CC(1/2) is correlation coefficient between shells of data, ideal number not clear- usually about 0.5 or higher, below 0.15 is non-significant correlation

Question 54

How is x-ray structure determination and refinement quality judged

Answer 54

R-factors Geometry Temperature factors

Question 55

R-factors

Answer 55

Sum of Fo-Fc / sum of Fo Dependent on geometry and completeness Rwork= crystallographic R factor where <15% excellent <20% good >20-23 ok, >25 means errors. Calculated with 95% of reflections which are used in carrying out refinement Rfree calculated with unbiased data 5% of reflections never used in refinement If both decrease, model is improving, if Rwork decreases and Rfree doesnt then model likely over-fitted

Question 56

geometry

Answer 56

How well structure fits expected structural properties of proteins Goal is bond length deviation <0.02 angstroms Goal bond angle <1.8% Must be restrained during refinement Effect of R-factor: if ignored then can make R-factor as low as desired See ramachandran plots

Question 57

How to check quality of model- quality assessment tools

Answer 57

Web based- PDB validation: Overall refinement metrics and Overall chain graphics Procheck

Question 58

What is the cry-EM revolution

Answer 58

Most rapidly developing technique in structural biology Growing field and features in major publications like Nature, Science and Cell each week Key starting point for vaccine and drug design Has been a huge increase in resolution due to improved detectors, microscopes, software and sample prep

Question 59

Steps in cryo-EM single particle reconstruction (SPR)

Answer 59

Sample preparation Sample freezing Screening and data collection 2D and 3D reconstruction Refinement and model building Interpretation

Question 60

How is cryo-EM sample prep done

Answer 60

By generating a higly pure and homogenous protein Good for membrane proteins as dont need a crystal Also by negative staining with heavy-containing atom stain- fast, easy, good overall view of protein and good as pre-cryo screening step

Question 61

How is cryo-EM sample freezing done

Answer 61

Dilute protein prepared in aqueous solution Frozen on carbon layer sitting on 3mm diameter copper grid Plunged into liquid ethane sitting in liquid nitrogen Having ideal sample prep hardest step Want water to freeze without ice as glass

Question 62

Cryo-EM screening and data collection

Answer 62

Want particles present in different orientations on EM grid to have all projections (wide distribution) Want good density on micrograph where individual particles are seen Want drift to be able to be well managed (apparent movement of specimen) Pick good particles, computer can align and see orientations and determine if all are there

Question 63

CryoEM 2D and 3D reconstruction

Answer 63

Projections can be simple or deceiving Need robust number of presentations and orientations for 2D construct to be made After picking those for 2D construction, they must be shifted, translated, rotated and paired. Then grouped and averaged (alignment) Simple objects can have complex projections 2D images become 2D classes 2D classes grouped into making 3D structure

Question 64

Refinement and model building in cryo-EM

Answer 64

Done on 3D classification, fine detailed improvements Can then build protein into the refined structure

Question 65

Why is structural biology research done

Answer 65

Starting point for biomedical research aimed at understanding molecular basis of disease Major jumping off point for experiments in drug and protein design Eg HIV protease structure was found, allowed to determine how it functions in disease and could make inhibitors based off of it