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Lecture 3
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1Why xray crystallography?
- to determine [blank_start]spatial[blank_end] arrangement of atoms (size, bond length, bond angle, bond type)
Define mechanistic details of binding and [blank_start]catalysis[blank_end]
Elucidate structural and [blank_start]functional[blank_end] relationship - have structure and use biochemical knowledge. [blank_start]NMR[blank_end] common technique to obtain structural info.
Structure-based drug design - method of designing medicines that relies on [blank_start]computational[blank_end] models of protein structures.- ANMR
- Bcomputational
- Cspatial
- Dfunctional
- Ecatalysis
2How to induce crystal formation.
[blank_start]Supersaturation[blank_end] - state of solution that contains more of the [blank_start]dissolved[blank_end] material than could be dissolved by the [blank_start]solvent[blank_end] under normal conditions.
Water evaporation
[blank_start]Nucleation[blank_end] - first step in formation. First contact that occurs between atoms that leads to further atom contact.
[blank_start]Crystal[blank_end] formation - drop in temp causes falling of solution in an ordered way to form a crystal.- ASupersaturation
- BNucleation
- CCrystal
- Ddissolved
- Esolvent
3Why is crystallisation of macromolecules difficult?
- complex, highly mobile surfaces, heterogeneity
- [blank_start]electrostatic[blank_end] nature - uneven [blank_start]charge[blank_end] distribution
- [blank_start]low[blank_end] chemical and [blank_start]physical[blank_end] stability - unfolding, hydration, temp <[blank_start]40[blank_end] proteins begin to lose structure
- overproduction, concentrating, purity
- specific factors - [blank_start]prostethic[blank_end] groups and ligands- Acharge
- B40
- Cphysical
- Dprostethic
- Eelectrostatic
- Flow
4Gene structure pipeline
Genomic info > Identification of [blank_start]genes[blank_end] > [blank_start]Cloning[blank_end] > Expression? [blank_start]Activity[blank_end]- Agenes
- BCloning
- CActivity
5Structural Genomics
Depend on [blank_start]expression[blank_end] [blank_start]vector[blank_end] choice
Aim to unravel structural functional relationship of all proteins encoded by [blank_start]genomes[blank_end]
They're high [blank_start]throughput[blank_end]
Limited by protein purification [blank_start]yields[blank_end]
[blank_start]Purification[blank_end] tag-position affects outcomes of process- Aexpression
- Bvector
- Cthroughput
- Dyields
- EPurification
- Fgenomes
6Important factors in crystallisation
- type of salt and/or salt [blank_start]conc[blank_end]
- type of salt and/or type of [blank_start]buffer[blank_end] pH
- [blank_start]colloidal[blank_end] properties of proteins, ionic strength and pH
- metal ions - solve [blank_start]phasing[blank_end] problems
- [blank_start]precipitant[blank_end] - some in orderly fashion, some completely [blank_start]amorphouss[blank_end]- Aconc
- Bcolloidal
- Cphasing
- Damorphouss
- Eprecipitant
- Fbuffer
7A crystal is a group of [blank_start]molecules[blank_end] arranged in a [blank_start]neat[blank_end] and orderly fashion. In an ideal crystal, content of each unit cell is [blank_start]identical[blank_end]. Precise stacking of unit cells allows crystal to have sharp [blank_start]edges[blank_end] and flat [blank_start]faces[blank_end].
A crystal can be thought of being made u p of many different sets of [blank_start]equidistant[blank_end] parallel planes passing through the lattice points - lattice planes. Many properties of the crystal can be understood in terms of spatial geometry of these planes. [blank_start]Miller[blank_end] [blank_start]indices[blank_end] are used to designate these different sets of planes.
How do we choose unit cell?
- [blank_start]simplest[blank_end] (smallest) repeating unit
- has to reflect [blank_start]symmetry[blank_end] of the crystal
-symmetrical operations are translation, rotation, reflection, inversion, glide- Amolecules
- Bneat
- Cidentical
- Dedges
- Efaces
- Fequidistant
- GMiller
- Hindices
- Isimplest
- Jsymmetry
8Precipitation zone - [blank_start]aggregation[blank_end]
[blank_start]Nucleation[blank_end] zone - crystalline form - encourages [blank_start]crystal[blank_end] formation
[blank_start]Metastable[blank_end] zone - [blank_start]supersaturation[blank_end] - can physically affect crystal formation- ANucleation
- BMetastable
- Csupersaturation
- Dcrystal
- Eaggregation
9Transitions in crystal formation:
Precipitate
Clear drop
Phase separatin
[blank_start]Granular[blank_end] precipitate
Spherulites
Cluster of [blank_start]needles[blank_end]
Plates
[blank_start]Microcrystals[blank_end]
Non-defined edges
Crystals from [blank_start]heavy[blank_end] precipitate
Crystals from granular precipitate
[blank_start]Optimised[blank_end] crystals- AGranular
- BMicrocrystals
- Cneedles
- Dheavy
- EOptimised
10Vapor diffusion set up:
[blank_start]Difference[blank_end] in precipitant concentration drives water evaporation. Protein goes from unsaturation to [blank_start]supersaturation[blank_end].
Hangin drop and sitting drop.
Precipitant solution contains precipitant ([blank_start]ammonium[blank_end] sulphate), salt (NaCl), [blank_start]buffer[blank_end] (Tris-HCl pH 7.5)
Volume of reservoir solution [blank_start]same[blank_end] as volume of protein
Concentration of reservoir is [blank_start]double[blank_end] conc of reservoir in drop so drives [blank_start]evaporation[blank_end], keeps evaporating until same conc, protein conc increases unsat to supersat.
If left for long, quality of crystal deteriorates.- Asame
- Bdouble
- CDifference
- Dbuffer
- Eammonium
- Fevaporation
- Gsupersaturation
11Cryoprotection
- reduces [blank_start]radiation[blank_end] damage
- traps [blank_start]protein[blank_end] chemistry
- crystals flash frozen in liquid nitrogen in a solution containing a [blank_start]cryoprotectant[blank_end] (glycerol) at 100k- Aradiation
- Bcryoprotectant
- Cprotein
12Lab sources:
- fixed [blank_start]wavelength[blank_end]
- low [blank_start]brightness[blank_end] - slow data collection
- xray beam exposes whole crystal
Synchrotron sources:
- tuneable wavelength ~[blank_start]1A[blank_end]
- very bright so [blank_start]fast[blank_end] data collection
- narrow beam so data collection from [blank_start]small[blank_end] crystals or small regions of large crystals
- high in [blank_start]cost[blank_end]- Awavelength
- Bbrightness
- C1A
- Dfast
- Esmall
- Fcost
Informações
- Descrição
- Biochemistry Quiz sobre Lecture 3, criado por Kate H em 01-04-2018.
- Autor
- Kate H
- Idioma
- English
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