Transcript Representations of 3D Structures

A b-strand is distinguished by strong CaHi-NHi+1contacts and
long range nOes connecting the strands.
A long range nOe connects residues more than 5 residues apart
in the chain.
A real example.
The rat fatty acid acyl carrier
protein. Involved in fatty acid
biosynthesis and part of a
larger subunit, the synthase,
Is it structured by itself??
Summary of the Sequential and Secondary NOEs observed for
rat FAS ACP - most definitely structured
NHi-NHi+1
aiNHi+1
biNHi+1
GDGEAQRDLVKAVAHILGIRDLAGINLDSSLADLGLDSLMGVEVR
D
D
D
DD
D
D
D
NHi-NHi+2
aHi-NHi+2
aHi-NHi+3
aHi-NHi+4
aHi-bHi+3
CSI
J
0-00000---------+-0-0--0+--+0+---+00+-0-----+ ++ -------+--+++++ +++-+++ --- ----- --QILEREHDLVLPIREVRQLTLRKLQEMSSKAGSDTELAAPKSKN
NHi-NHi+1
aiNHi+1
biNHi+1
D
D
D
D
DD D
D
D DD
NHi-NHi+2
aHi-NHi+2
aHi-NHi+3
aHi-NHi+4
aHi-bHi+3
CSI
J
-----0+-+0++--0--00+--------00000000+0+00-00
-++-+++ - -- -+-+ - -+- +++++++
+++
So I have assigned the NMR spectrum and connected the
amino acids. I have a good idea of the secondary structure.
What next??
At this point we notice there are still many nOes we have not assigned on
the 2D spectrum. These are neither sequential or short range. They are
long-range and connect residues more more than 5 amino acids apart (But
still close in space!). O
H
N
CH
O
C
H
N
CH2
Asn
C
CH
C
Gly
H
O
N
H
H
OH
Identified as an
asparagine aminohydrogen from COSY
spectra
HO
O
C
CH2
Glu
H2C
C
O
CH
NH2
NOE indicated the
asparagine amino-hydrogen
is near a glutamate acidic
hydrogen
Schematic showing long range nOes in the lac headpiece protein
What next? STRUCTURE CALCULATIONS
•From NOE I know close atom-atom distances, but that doesn’t give a structure
•The information you have up to this stage is a list of distance constraints
•The structure can be determined by inputting this information to computer
minimization software.
•The computer program also contains information about amino acids, bond
lengths/angles and standard information about atom-atom interactions such as
minimum distance (i.e. Van der Waals radii)
•With all this information you can generate a model of the structure.
Important: NMR gives you a number of possible solutions
(all almost identical, rmsd <1Å), This can range from 5-20 models
X-ray crystallography give one average structure
NMR structures can be averaged to give one average structure as well
The power of the NOESY experiment is that the intensity of an
NOE peak will be related to the nuclear separation.
Strong NOE crosspeaks - 1.8-2.5 Å
Medium NOE crosspeaks – 1.8-3.5 Å
Weak
NOE crosspeaks – 1.8-5.0Å
Excerpt from an NOE table for Actinorhodin Polyketide ACP - 1997
This file contained ~ 700 lines of nOe restraints
! Thr7 NH
assign (resid
7 and name HN
)(resid
75 and name HD1* ) 4.0 2.2 0.5
assign (resid
7 and name HN
)(resid
75 and name HD2* ) 4.0 2.2 0.5
assign (resid
1 0 and name HN
)(resid
75 and name HD2* ) 5.0 3.2 0.5
assign (resid
1 0 and name HN
)(resid
75 and name HD1* ) 3.3 1.5 1.0
assign (resid
7 2 and name HN
)(resid
31 and name HD1* ) 5.0 5.0 0.5
assign (resid
7 2 and name HN
)(resid
31 and name HD2* ) 3.3 1.5 0.5
assign (resid
7 2 and name HN
)(resid
31 and name HB*
) 4.0 4.0 1.5
assign (resid
7 2 and name HN
)(resid
31 and name HA
) 4.0 4.0 1.0
7 5 and name HN
)(resid
10 and name HD1* ) 4.5 4.5 1.0
! Leu10 NH
!Arg72 NH
! Leu 75 NH
assign (resid
The simulated annealing protocol. Begin by simulating a 1000K heat bath and
generate an extended model strand along x (random coordinates along z,y)
Start
x
Apply the distance restraints from the NOE data (perhaps 1000 restraints for
a protein of 90 amino acids). Weight the nOes to favour the formation of
local secondary structure and later long range structure. Allow chain to
move through itself by reducing the effective Van Der Waals radii
30 ps
Start to cool the system and increase the penalty for not satisfying an NOE.
20 ps
Minimize the final structure to see if it satisfies all the nOes
A simulated annealing trajectory over the first few picoseconds
4 helices begin to
‘condense’
Unfolded
Correctly folded
Challenges for Interpreting
3D Structures
• To correctly represent a structure (not a
model), the uncertainty in each atomic
coordinate must be shown
• Polypeptides are dynamic and therefore
occupy more than one conformation
– Which is the biologically relevant one?
Representation of Structure
Conformational Ensemble
Neither crystal nor
solution structures
can be properly
represented by a
single conformation
 Intrinsic motions
 Imperfect data
Uncertainty
RMSD of the ensemble
Representations of 3D
Structures
C
N
Precision is not Accuracy
These 2D methods work for proteins up to about 100 amino acids,
and even here, anything from 50-100 amino acids is difficult.
We need to reduce the complexity of these 2D spectra.
1
16
1
Ha
R2
O
HN
16
O
12
Ca
12
C
14
N
14
N
12
C
12
Ca
1
R1
1
Ha
HN
We can start by
replacing 14N with
15N, a spin 1/2
nucleus.
HSQC of rat FAS ACP
15N shift of nitrogen of amide bond
1H-15N
H
N
1H
Chemical Shift
X
89!