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X-ray Crystallography
Austin Schwartz and Kassi Ferguson
What is X-ray Crystallography?
http://www.theguardian.com/science/video/2
013/oct/09/100-years-x-ray-crystallographyvideo-animation
X-Ray Crystal Structures
• Provide structural insights into
protein mechanism at the atomic
level
• Mean resolution = 2 Angstrom (0.2
nm)
• Membrane proteins are
notoriously difficult to crystalize
• Obtaining novel crystal structures
is generally a long process that
includes many strategies that do
not work
• No “one size fits all” strategy for
crystallization (however GPCR
structures are becoming more
standardized)
GPCR opsin, Park et al. 2008
What is X-ray Crystallography?
• Used to determine the
arrangement of atoms
within a crystal at high
resolution
• A beam of X-rays strikes
the crystal and diffracts
into many specific
directions
What is X-ray Crystallography?
• Based on the angles and
intensities of the
diffracted beam, a three
dimensional structure of
the density of electrons
can be determined
• From here, the mean
positions and chemical
bonds of the atoms
within the crystal can be
determined using
mathematical Fourier
transforms
Crystal Growth
• With purified protein…
–
–
–
–
–
Cooling
Evaporation
Vapor Diffusion
Liquid Diffusion
Sublimation
Crystals are typically larger than 0.1 mm
Membrane Proteins
• Insertion of membrane proteins into the plasma membrane is driven by
favorable helix-lipid interactions
• Some polar transmembrane segments require help by neighboring
proteins
• Protein is not fully folded until it is inserted into the membrane
• 75% of membrane proteins have a intracellular N-terminus
• N-terminal tags aid in purification and do not hinder folding
• Tags on extracellular N-terminus hinders translocation into
membrane
Detergents for Membrane Proteins
• During purification, detergents are used to solubilize
membrane proteins
• Contain polar head group and hydrophobic tail, just like
phospholipid bilayer
Detergents for Membrane Proteins
• During purification, detergents are used to
solubilize membrane proteins
Conventional Detergents, with polar head
groups on right and hydrophobic chains
at left
Novel Detergents, amphiphiles contains
“side” polar groups
Stability in Detergent
Stabilizing Membrane Proteins
• Point mutations
• Example (Warne et al. and SerranoVega et al., 2008)
– Alanine scanning
mutagenesis used to
stabilize the turkey B1adrenergic receptor
– Every residue was
substituted with alanine
and the effect of antagonist
binding was measured at
elevated temperatures to
determine stabile mutant
• Computational methods
Stabilizing Membrane Proteins
• Membrane protein scaffolds
• Fab fragments from monoclonal antibodies
KvAP Crystal Structure stabilized by
FAB fragments on the E2
Extracellular loop, Jiang et al 2003
FAB fragment
KvAP
Types of Crystals
• Type 1: 2D crystals with each membrane protein
oriented side by side with hydrophobic surface of the
protein initiating crystal contacts
– Prepared in a lipid environment
– Difficult to make
• Type 2: Crystal contacts formed by the hydrophilic
surface of membrane proteins, while the hydrophobic
portions are shielded by detergent
– Prepared in a detergent environment
– Easier to make
– The majority of membrane proteins have be determined
with type 2 crystals.
Disadvantages for Membrane Proteins
• Difficult to obtain stable
membrane proteins in
crystals without mutating the
protein
• Not in a native lipid bilayer,
thus not a true native
conformation
• Crystal packing can include
non-native conformation
Structural Variability at Neutral pH
Neutral pH
pH 4 (open)
Fluctuations in Crystal Structure
Neutral pH
pH 4 (open)
Changes in Functional Aspects of ECD
Neutral pH
pH 4 (open)
Changes in Functional Aspects of ECD
ECD-TMD Interactions
Open and Closed forms Coexist at pH 4
Overview
Neutral pH
pH 4 (open)
Kv1.2
• Voltage-dependent K+ channel
• Member of the Shaker Kv
Family
• Contain highly conserved ProX-Pro signature sequence
• Preceding the N-terminus,
Shaker channels form a T1
domain
• Beta2 subunit docks at the T1
domain
– Contains an active site for
NADP+
– Function not known,
potentially regulates Kv
activity
Kv1.2 Expression
• Kv1.2 from rat was coexpressed with the B2 K
channel beta subunit, in
yeast Pichia pastoris
• Channel contained a Nterminal 8x His tag and
TEV protease cleavage site
• N207Q mutation to
elimination glycosylation
at the S1-S2 linker
– Preventing glycosylation
may play reduce trafficking
to membrane
Pichia pastoris
To form Kv1.2 Crystals…
• Protein was kept in a mixture of detergent and
lipids
– N-dodecyl-B-D-maltopyranoside or n-decyl-B-Dmaltopyranoside) and 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine and 1-palmitoyl-2oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
• Channel was kept in a strongly reducing
environment using DTT and TCEP and in a
oxygen depleted atmosphere
Crystals Formed via…
• Vapor diffusion and frozen in liquid nitrogen
Kv1.2 Electron Density
Electron Density Map = Blue
VS = Voltage Sensor
T1 = T1 Domain
B = Beta Subunit
T1 and Beta Subunit are more
electron dense than pore and VS
Mean B Factor
T1 and B = 59 A2
Pore = 159 A2
VS = 162 A2
Based on electron density, a model
of the channel was made
Kv1.2 Crystal Lattice
Pore and VS (membrane
spanning) = Red
T1 and Beta Subunit
(extramembranous regions)
= Blue
Due to low electron density,
VS model built without
connecting loops S1-S2, S2S3 and S3-S4. S4-S5 was
included
The Voltage Gated Potassium Channel
Extracellular
Intracellular
Kv1.2 Crystal Structure
VS
135 A
NADP+
95 A
95 A
Pore
S4-S5 linker
Kv1.2 Crystal Structure
Open Pore
conformation
VS
12 A
Pore
S4-S5 linker
Intracellular
Extracellular
Comparison of Kv1.2 to other K+
Channels
Kv1.2 = Red (open pore)
KcsA = Gray (closed pore)
KvAP = Blue (open pore)
* = Pro-Val-Pro
Charge Mapping of Kv1.2
Glutamate and aspartate residues = (-)
charge = Red
Arginine and lysine = (+) charge = Blue
* = (-) charged Glu 128, Asp 129 and Glu
130 on T1 domain that interacts with
positively charged amino acids on
inactivation peptides for N-type
inactivation (ball in chain)
Virtual Drug Screen
• Exploit differences in GluN1 and GluN3
orthosteric binding site
• Virtual screen of GluN3A LBD in antagonist
bound state
• 99 compounds obtained and functionally
screened at GluN3A/B
• Active hits evaluated at GluN1/2
• TK40 
TK40 Selectively Binds GluN1
Antagonist Drug Potency Over Agonist
100 mM glutamate
0.5 mM glycine OR
100 mM glycine alone
3 mM glutamate
3000 mM glycine OR
10 mM glutamate alone
TK40 Affinity for GluN1
Radioligand Binding of TK40 to GluN1
TK40 Effect on GluN3A vs GluN3B
Crystal Structure: GluN1 with TK40
Bound
Residues for GluN1 Specificity