Download Proteins - Structure, folding and domains

Proteins - Structure,
folding and domains
Tommi Kajander
X-ray crystallography lab
Institute of Biotechnology
University of Helsinki
Outline
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basics on proteins
stability and folding
structures and domains
X-ray crystallography
protein structure
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primary structure, amino
acid sequence, the peptide
bond, polypeptide runs from
amino(N) to carboxy(C)terminus of the chain.
secondary structure, tertiary
structure (the folded state)
+ oligomeric state
(quaternary structure).
cellular functions
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produced by living cells, translated from
the encoding gene.
may function inside the cell (in its various
compartments, e.g. nucleus -DNA binding
proteins- or cytosol or on the cell
membrane or be secreted out (e.g.
microbial enzymes such as cellulases etc,
animals growth factors, antibodies,
lysozyme, proteases etc (food processing in
the stomach, gut).
Protein functional classes
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enzymes
structural proteins (collagen etc)
ion channels
transporters (hemoglobin, throughmembrane transport)
immune system proteins (binding)
other binding proteins (growth factors,
DNA binding proteins, chaperones)
often functionalities are specific to specific
domains (see below)
Building blocks
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the 20 natural Lamino acids (no Damino acids)
the peptide bond
amino and carboxytermini, direction of
the polypeptide
zwitterions, chiral
with different “side
chains”
The 20 amino acids
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20 natural amino acids encoded by DNA (one
amino acid per base triplet, 64 triplets)
all L-amino acids (stereochemistry), chiral
around Cα−atom.
hydrophobic and polar and charged.
what does this mean, implications? learn them!
some with special properties (Gly, Pro, Cys..).
atom nomenclature:
– heavy atoms: N,C,O, Cα (peptide unit) + side chain
atoms Cβ, Cγ, Xδ etc with distance from Cα.
– some side chains are branched (numbering 1,2..)
– three letter and one letter codes (Glysine, Gly, G)
hydrophobic amino acids
inside proteins
„ the hydrophobic core,
sticky binding pockets.
„ aromatic rings,
stacking. delocalization
of the π-electrons
(Tyr, Trp, hydrogen
bonds)
„ volume and shape
„ Proline ring, imino acid
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polar and charged
Sulphur containing amino acids
• Cysteine and methionine
• Cys the most reactive amino acid,
thiol group can oxidize and
deprotonate, pKa ca 8-9.
• disulhide brigdes, structurally
important. also non-spesific
dimerization/aggregation via free
-SH groups
•Met potentially nucleophilic,
hydrophobic.
Acidic groups
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Aps, Glu.
pKa = 4 can vary with environment
substantially (proteins tune the
functional group protonations)
active sites/enzymes
metal ion binding (also other polar
residues (e.g His and transition
metals)
acid/base catalysis
thermostability/ionic networks
Basic groups: Arg, Lys,
His
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positively charged under
physiological pH (neutral)
His, metal coordination and
catalysis
pK around 6-7, protonation state
variable (mostly not charged?)
Lys, catalytic base in active sites
Arg, ion-pairs, electrostatic
interactions in active sites and
ligand (e.g PO4) binding
Other polar
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Asn, Gln
– hyrdogen bonding, Asn
can deaminate.
– Asn, N-linked
glycosylation of proteins
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Ser, Thr, Tyr
– H-bonds, Ser in catalysi,
Ser, Thr, Tyr are
phosporylated and
dephosphorylated in
cellular signalling events
(most common
signalling method)
“turn” residues
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Glysine, Proline
– common in turns, break secondary structure (not always)
– Pro is an imino acid, generates kinks, if not turns.
– both destablize secondary structure in any case (helices and
strands)
– Pro and hydroxyl-Pro in collagen triple helix (also Gly).
– Gly has NO side chain.
– Pro is really an imino acid cis or trans form,
usually trans. Pro is rigid, Gly can have variable peptide bond
phi and psi angles.
pKa, pI
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pKa -log10 of the acid dissociation constant:
– Ka = [H+][A-]/[HA] for HA <-> H+ + A-
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pI, the isoelectric point, net charge of protein is zero.
– can be calculated from sequence (google protparam),
approximately, or experimentally ,by isoelectric focussing.
– so this tells whether protein has + or - charge at specific pH.
– implications on solubility?
pKas of amino acids
residue
Asp
Glu
His
Cys
Tyr
Lys
Arg
pKa
4.5
4.6
6.2
9.1-9.5
9.7
10.4
ca. 12
physiological pH (7.4)
charged(-)
neutral/charged
-SH(neutr.)
-NH3+ charged(+)
charged(+)
roles of residues in
proteins
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hydrophilic out, hydrophobic in (or more
precisely the groups not necessarily whole side
chains, e.g. lysine has a long aliphatic
hydrocarbon chain)
functional/active site specificity is in shape
(conformation and hydrophobic) hydrogen
bonding (polar/charged)
catalytic residues (acids, bases, nucleophiles,
metal ion binding)
about enzymes
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types of catalysis:
– general acid/base catalysis (Lys, Asp, Glu, His)
– electrostatic catalysis (exclusion of solvent)
– nucleophilic and electrophilic catalysis
Ser, Cys (proteases) most common
nucleophiles, metals as electrophiles
(+ Reduction of entropy on binding, high effective
concentration, catalysis in preordered active
site)
The Serine protease catalytic triad (electron transfer chain)
amino acids in the
polypeptide context
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polypeptide backbone + side chains of
the amino acid residues = protein
protein synthesis
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peptide bonds are formed (in cells) on the
ribosome on translation of the of
messengerRNA(mRNA), amino acid gets
transfered from the transferRNA(tRNA) and
added to the polypeptide C-terminus.
Structure formation
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properties of the side chains determine the higher order
structure of proteins, and functionality, mostly.
hydrogen bonds between and from the peptide “backbone”
amide and carbonyl groups are important for secondary
structure (still defined by side chains)
peptide bond is planar (important!)
proteins can be a) fibrious/filamentous (collagen, silk,
muscles myosin and actin), b) soluble (e.g. enzymes, growth
factors, insulin etc etc), or in the cell lipid membrane c)
“membrane proteins” (hydrophobic/lipid soluble) (ion
channels and pumps, control of cell homeostatis, transporters
and receptors (G-protein coupled receptors, signalling),
typically cell surface receptors have a intramembrane domain
+ extracellular ligand binding domain + intracellular region
for signalling inside the cell.
Secondary structure
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fold/structure of the protein stabilized in
secondary structure, α-helices or β-strands
sequencial arrangement (topology) and
spacial organization of these elements
defines the FOLD of the protein
amino acids have different propensities for
forming particular secondary structure (e.g.
Ala and non-β branced residues in α-helices
steric restraints
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Ramachandran plot of polypeptide
main chain variable angles (phi, psi)
only a restricted set of conformations
available for different secondary
structures (and amino acids)
steric effects, due to side chains -->
Gly has most freedom, hence
important in turns.
polypeptide “backbone”
angles
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3 main chain
dihedrals
Only two really vary
– Create all of the
backbone structures
seen in proteins.
Ramachandran plot
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Beta strands - spread out
Alpha (lh) helices mostly glycine
„ Alpha (rh) helices bunched up
„ >90% of residues should
be in most favoured
areas
„ Glycines in many areas
from
MLE, code 1MUC
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α-helix
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3.6 residues/turn
main chain h-bonding
from i to i+4.
1.5 Å rise/residue
5.4 Å rise/turn
Independent
stabilization by Hbonding.
Sidechains point back
like a Christmas tree.
helices often have
different faces
helix dipole/capping
(N/C)
helical wheel and packing
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amphilicity, binary pattern (polar/aliphatic,
helical packing), “heptad repeat”
packing: “grooves to ridges” (in real life more
variable, Bowie Nat. Struct. Biol. 1997)
β-strand
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Flat-ish
Two varieties
– parallel
– antiparallel
Often distorted
– Twisted, Bulged
Certain proteins can also
form amyloid fibrils
=“infinite Beta-sheets”
(Altzheimers’s, prions,
other amyloid diseases,
e.g. lysozyme and betamicro-globulin and
transerythrin variants)
antiparallel β-sheet
β-turns
• nearly 1/3 of residues involved
in turns (various types)
•hairpin loops, connect betastrands
• classification (here, type I and II),
torsion angles
Supersecondary structure
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Regular secondary
structure patterns
– Sometimes
functional
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HTH motif (DNA
binding element)
– Sometimes
structural
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β−α − β
β−hairpin
sequence analysis
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conserved domains or motifs (web tools).
membrane vs. soluble proteins
– hydrophobicity plots(scales), again the www.
– types of membrane proteins: (alpha-helical e.g.
7-TM proteins, ion-channels etc), beta-barrel,
single-span alpha-helical (many receptors and
adhesion molecules).
GABA-transporter
domains..
Domains
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Defined as independently-foldable units.
Often mark different functional units.
Separated by linker regions.
MLE, code 1MUC
Goldman, Helin et al.
Domains
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Mammalian proteins
often are “beads on
a string”
– Repeated domains
often occur
– Different functions,
structural variations
– See e.g. SMART or
PFAM databases
(Google) for domain
compositions of
proteins
Domains 19-20 of Factor H
Jokiranta et al & Goldman, EMBO J,
2006
Proteins Folds
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Some protein folds are unique, but the “fold-space”
must be limited (structural genomics attemps to
map this).
many proteins have several domains
Most proteins fold into already-known structures
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Some common folds
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– Even when there is no sequence homology (<10-20%)
– Structure preserved much longer than sequence.
– β/α barrel
– 7-TM receptor
– 4-helix bundle
− β-sanwich domains (immunoglobulin-like) etc.
β/α barrel
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β/α barrel
– 40% of unique
proteins
– Usually enzymes
– 8 repeated β/α units
7-TM fold
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7-TM fold
– Example is Bovine
rhodopsin
– >500 such folds in
humans
– 7 transmembrane
helices
– Signal transduction
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signals very varied
PDB code: 1F88
Repeat proteins
Tetratricopeptide
Repeat (TPR)
Leucine rich repeat
HEAT repeat
Ankyrin repeat
-protein-protein interactions
in various cellular contexts
-structural scaffolds
The β-propeller (a repeat
protein of sorts)
-All β-sheet
-4-7 ”blades” with 4 strands
-Neuraminidase,
WD-40 repeat proteins
(Gproteins).
-binding site often
in the center
β-structures:
”Sandwich”, barrels,
propellers
Neurospora crassa CMLE, Kajander et al. (2002)
The β−sandwich fold
-Antibodies (Immunoglobulins), adhesion molecular and receptors
NCAM, ICAM etc, RAGE, FGFR etc.
Antibody structure
multidomain structure..
integrin αvβ3
Hsp90 complex
with p23
protein complexes
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Stable
– Ribosomes (small and large subunit) (proteinRNA)
– FoF1 ATPase (ATP synthase, proton pump)
– Proteosome, etc etc.
Transient
– Nucleic acid polymerase complexes (proteinDNA)
– chaperones and their (unfolded) substrate
proteis
– signalling complexes (e.g G-proteins and their
receptors)
viral capsids
Protein folding
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“the protein folding problem”
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complex, vs e.g. DNA/RNA structure
Can’t predict the structure ab initio
but there are cases of success for small
proteins
what stabilises the folded (native) state
driving forces
mechanisms
protein folding
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Levinthal’s paradox
– folding via sampling all conformations (contact order) would take
more than age of the universe --> folding landscape/not all conformations
are explored.
– Original example: 3100 = 5 X 1047, 1013 per second, or 3 x 1020 per year, it
will take 1027 years….
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hydrophobic vs hydrophilic:
– the most basic idea of protein folding is binary patterning
(used in protein design of helical bundles, most simple case).
Kamtekar et al & , Hecht MH. Protein design by binary
patterning of polar and nonpolar amino acids. Science (1993)
folded/native state
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free energy minimum, typical
functional proteins have a single
defined native state
hydrophobic and aromatic residues
inside
charged/polar residues on the outside
OR hydrogen bonded (solvation by
the protein)
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hydrophobic and aromatic residues inside
charged/polar residues on the outside OR hydrogen
bonded (solvation by the protein)
– one observed feature of thermophilic proteins is increased
number and extent of ionic networks
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minimal “alphabet” designs (polar, hydrophobic, turn, e.g
funcitonal SH3 domain by Riddle et al & Baker D. amino
acids: Ile, Lys, Glu, Ala, Gly)
Both cases used combinatorial methods (selection or
screening) from mutant libraries (Kamtekar et al & Riddle
et al ...and others)
Thermodynamics of
folding
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large opposing and favoring terms
– the hyrdophobic effect (release of water from hydrophobic
surface, entropy driven)
– hydrogen bonding of the backbone, favorable enthalpy
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Cooperativity, e.g. helix and beta-sheet formation, main chain hydrogen bonds
define folded topology.
all buried side chains solvated by H-bonding
– loss of conformational freedom of the protein chain
(unfavorable entropy)
– other effects? charge burial? small proteins <100 residues,
sometimes stabilized by bound metals or disulphides.
„ Substratcting two large values: proteins are marginally
stable (ca. 5-20 kcal/mol) ...and one hydrogen bond is (2-10
kcal/mol)
Thermodynamic studies
of protein stability
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denaturation by heat or chemical
denaturation 0-6 M Gu-HCl
CD spectroscopy (secondary structure)
Trp-fluorescence quenching (tertiary
structure)
Protein folding
CI2: collapse & formation of secondary structure simultaneously:
nucleation-condensation (a basic foldon)
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typically single domain proteins exhibit two-state unfolding with sharp
transition
multidomain or oligomeric proteins not.
SPECIAL CASE (?): repeat proteins: The 1-D Ising model and
mechanical unfolding (step-wise + elasticitity, hearing (hair cells in ear)
AFM
1D-Ising model: Stability of TPRs vs. number of TPR repeats
• a two-state transition
• Increase in stability with increasing number
of repeats
• Increase in cooperativity (slope of transition)
with number of repeats
(in addition to dominant local interactions and
regular structure retained as repeats are added)
The 1D-Ising model captures this behavior (as shown for helix-coil
transition by the Zimm-Bragg model):
=(spin up, +1) folded helix
=(spin down, -1) unfolded helix
= description of folded/unfolded helices (in TPRs)
Æ each “spin” up-down interaction
Kajander et al. & Regan (2005) J. Am. Chem. Soc.
will cost energy
Folding states
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Equilbr. U->N, U->I->N (end states
observable)
Seen by several transitions, or
difference in fluorescence and far-UV
CD-spectroscopy
Kinetics tell about the folding pathway
Folding kinetics
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Two-state kinetics vs intermediates
First-order kinetics if 2-state
Transtion state theory:
– kf=υκ exp –∆G‡/RT
Æ ∆∆G‡=RTln kf’/kf
Chevron plots
• measure folding & unfolding (dilution to or from e.g. GuHCl)
• deviations from: effects of intermediates, mutations / what they affect.
Folding, φ-value analysis
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in order to study the folding pathway one needs to look at kinetics (e.g. trpfluorescence by stopped-flow rapid mixing)
e.g. phi-value analysis of mutants (Ferhst & co-workers)
Range from 0 to 1 (effect of mutation on denatured or folded state).
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0=(already) unfolded in transition state (mut: no effect), 1=structured in
transition state.
Mutate away
H/D exchange with NMR.
ÆVariability in local stability
protection factor (pf) = ku/kex
CTPR2 & CTPR3 –NH hydrogen exchange (Main et al. & Regan PNAS 2005)
3D-Structural Techniques
of biomolecules
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X-ray (and neutron) scattering & crystallography.
– Elucidates structure-function relationships at the atomic level.
– Very large size range (40 Da to > 1 MDa) (ribosome, viruses)
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NMR spectroscopy also provides detailed structural data.
– Complementary to crystallographic efforts and results.
– NMR has been restricted to molecules below 40 kDa.
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Electron microscopy, single particle and diffraction.
– Low resolution, but highest molecular mass (eg Clathrin pits,
whole ribosome, enveloped viruses)
Strategy in protein structure
determination via X-ray
crystallography
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Obtain pure preparations in milligram quantities
of the macromolecule(s) of interest.
Screen for preliminary crystallization conditions
on an incomplete factorial basis. Consider the
biochemistry.
Refine crystallization conditions / Mutants /
protein chemical modifications.
Collect diffraction data from suitable crystals.
Solve the “Phase” problem.
Analyze the electron density maps and build the
atomic model.
Landscape of Structural Biology
Cloning
Model Building
and Refinement
Protein Expression
and Purification
Crystallization Trials
Data Collection
Optimization
crystallization
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typically by vapour
diffusion
also microdialysis or
“batch” (no diffusion).
or diffusion in a
capillary
use 24/48/96 wellplates
different temperatures
(4/RT/other)
start with various
random screens
Crystallisation automatin with
robotics
http://www.biocenter.helsinki.fi/bi/xray/automation/
Crystals
Crystals and Symmetry
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Why do we need a
crystal?
– Scattering from a
single molecule is
undetectable.
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need a lattice
– Crystals have
translational
symmetry
– Can resolve features
at atomic resolution.
Unit Cell
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Smallest object from
which you can make
the entire crystal by
translation along the
edges.
Bounded by lattice
points.
Edges: a, b, c
Angles: α, β, γ
What is in a unit cell?
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A unit cell is built from asymmetric units.
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Asymmetric unit
L
L
L
L
L
L
L
L
L
L
L
L
L
L
Examples
L
L
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L
– the smallest unit from which a unit cell can be
built by application of crystallographic
symmetry operators.
L
Translational Symmetry
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Cover space
– Screw axes
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sliding rotations
21, 31, 32, 41, 42, 43
repeats that extend
across unit cells
– (Glide planes)
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sliding mirrors
21
41
42
Asymmetric unit
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The smallest object from which the unit cell can be
built up, by application of crystallographic
symmetry.
Contents of an asymmetric unit
– At least one macromolecule per asymmetric unit.
– Can be more - if there is non-crystallographic symmetry:
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a dimeric protein may be in the asymmetric unit (or not).
the 5-fold symmetric virus coat is always in the asymmetric
unit.
Symmetry
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Crystallographic (Space group) symmetry
– The symmetry elements apply throughout the
crystal.
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A 2-fold axis in unit cell A will be coincident with other
2-fold axes in different unit cells.
Non-crystallographic (local) symmetry
– The symmetry elements apply locally only to the
atoms around a particular lattice point.
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Axes are parallel but not (except by chance) coincident
with each other.
Crystal
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Crystal lattice
– Asymmetric unit + symmetry = unit cell
2
What is a protein
crystal?
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Crystal lattice is a periodic, symmetric system:
– Proteins are asymmetric and fit periodicity poorly
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Inorganic crystals are hard and dry
– protein crystals are moist and include up to 70%
solvent .
– Solvent channels between molecules:
– Possible to soak in chemicals; enzymes can be active
in the crystal
Bravais lattices
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Bravais, 1850:
– 14 different crystal lattices
possible:
– Due to symmetry of unit cell.
– Constraints on cell lengths &
angles.
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230 space groups; 65
biological ones
Point group
– Symmetry around a point.
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Space group
– Symmetry arrangement that
covers space.
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The word “group” here has its
full mathematical meaning.
Space Groups
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Certain space groups are more common in
protein (and other) crystals.
60% of the PDB structures belong to 5
space groups:
Space Group
Percentage (1997)
P212121
23 %
P3221
8%
C2
8%
P21
11 %
P21212
8%
Space groups
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230 possible space
groups
– All available symmetry
operations on all 14
Bravais lattices.
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Biological ones are
chiral
– 65 of those.
Protein crystals
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Only limited amount of symmetries are allowed
for proteins: no mirror!
Symmetry has to fill the space: 2-fold, 3-fold,
4-fold, 6-fold (no 5 or 7 fold etc!! Can’t build a
crystal lattice!!)
Screw axis
– Ex. P21
Crystals are not like
solution?
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Protein crystals
containing 35-75%
(vol.) water
Crystals can be active
Enzyme activity
Time-resolved
crystallography
Bacteriorhodopsin
photobleaching
– Pumping in the D96N
variant crystal (colour
changes)
Myoglobin CO photolysis
Diffraction
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Are these good ?
For our purposes
they are good
when they diffract
X-rays well.
Data collection
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Data from a synchrotron
Microscopy
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How it works
– Specimen
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diffracts parallel light
– objective lens
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focusses diffracted
image
Diffraction
different orders
of diffraction (waves)
1,2,3,4,5....
Crystal Lattice
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A theoretical
concept
Lattice planes (Hila
tasot)
– Can be defined by
their intersection
with the axes
– Spacing, d, between
the planes used in
Bragg’s law.
in phase from identical lattice points
Braggs law:
nλ=2d sinθ
Fourier transform
• adding up the components
of nth (of increasing
frequency) order will give the
sum = repeating unit.
• the more terms the finer the
detail
ρ xyz
1
=
V
∑∑∑ | F |
h
k
Measured
hkl
eiα e − 2πi ( hx + ky +lz )
l
Phase
Fourier Transform
The “phase problem”
?
Obtaining the phase(s)
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to reconstruct the diffracted waves need to know
the phase, intensities collected from diffraction
give amplitude (of the wave) I = |F|2
Only the Intensities can be directly obtained by
the X-ray detector.
patterson function
•methods (in practise):
•heavy atom derivatization, inclusion of (other)
anomalous scattering elements (e.g. SelenoMethioninelabeling, Br-nucleotides), or using a model (homologous
(ca. >30-40 % id.) structure) and find the position and
orientation of this in the crystal unit cell (asymmetric unit
more exactly).
•Find the heavy atom structure with patterson function
calculation
(a fourier transform without the phases)
1
−2 πi(hx +ky +lz )
Phkl = ∑ ∑ ∑ Ihkl e
V h k l
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Difference patterson function
(IPH-IP=IH)
Cross vector peaks between
atoms related by symmetry
operators of the space group
found on special sections called
the harker planes.
From this can be obtained
H (x,y,z)
Structure solution
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Obtain initial electron density with the heavy
atom coordinates (initial phase refinement)
Build a model (and/or use non-crystallographic
symmetry averaging and solvent flattening to
improve maps, possibly phase extension to
higher resolution if data available)
Automated chain tracing programs + hand
building on graphics
A map phased with 4 Ptatoms
finishing the structure refinement
fit model back to
X-ray data via a
minimization
target function
(minimize an
energy, including
known
geometric/chemical
terms)
The meaning of resolution (1.92.5-3.0Å)
lattice plane separation d, smaller the separation the wider the diffraction angle
= higher resolution of “image”
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How do you know when the crystal structure is
right?
– Data completeness,
– signal/noise (I/sigma)
True measures of resolution
– R-factor (Rwork, Rfree)
R=
– Geometry (Ramachandran plot,
r.m.s.d. of bonds and angles)
– look at the electron density maps
∑F
o
− Fc
∑F
o
Links and literature
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Google: PDB, www.expasy.com (various seq. analysis tools +
swissmodel), SMART, PFAM + web courses on crystallography.
EDS: http://eds.bmc.uu.se/eds/ (view maps for PDB structures)
(protein interaction domains:
http://pawsonlab.mshri.on.ca/index.php, doenst include all...)
Free PC/Mac sofware for structure viewing and analysis: Pymol
(best by far), Swiss-PDBviewer, Rasmol (see also PDB site).
Reading
– Introduction to protein structure, Bränden C and Tooze J.
– Crystallography made crystal clear, Rhodes G.
– Outline of Crystallography for Biologists, Blow D.
– Structure and mechanism in Protein Science, Fersht A.