SAXS and SANS Facilities and Experiments

velodromeryeUrban and Civil

Nov 15, 2013 (3 years and 10 months ago)

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Jill
Trewhella
, The University of Sydney

EMBO Global Exchange Lecture Course

April 30, 2011

SAXS and SANS Facilities and Experiments

The small
-
angle scattering experiment

FT

X
-
ray Sources


CuK


(1.54 Å) emission from


Sealed X
-
ray tubes, practically need a line
source geometry for protein work


Rotating Anode source, allows for point source
geometry and hence simpler analysis


Synchrotrons


Tunable (anomalous scattering)


High Brilliance


Excellent for rapid scanning of conditions, very
low protein concentrations, time resolved
experiments, etc


Lab
-
based

X
-
ray sources

Point versus line collimation

Slit ‘smearing’

Anton
Paar

SAXSess

(line source)
at the University of Sydney


X
-
ray scattering samples


5
-
60 minute measurement times using lab based
sources


Protein concentrations 1
-
10 mg/ml


Sample volumes in the order of 20
-
30 µL


Experiments using high intensity synchrotron
instrumentation take a few seconds or minutes and
particle concentrations can be more dilute (by at least an
order of magnitude), but radiation damage can be
limiting; free radical scavengers are helpful (DTT, TCEP,
ascorbate)


Need a perfectly matched solvent blank; preferably a last
step dialysate or column filtrate


Sample cells are made of ultra
-
thin quartz or mica, must
be able to measure sample and solvent background in
same cell, identically positioned in the beam

Neutron Sources


SANS requires ‘cold’ source; thermal neutrons
are passed through a liquid hydrogen
moderator to slow them (generally to ~4
-
6 Å)


Reactors are steady state sources of cold neutrons
that are collimated to provide a narrow wavelength
band (

/


~10%)


Spallation sources produce pulses of cold neutrons
and instruments are designed to use time of flight
so that all wavelengths in a given pulse can be
used which compensates at least partially for
relatively the low time
-
averaged neutron fluxes

Recall: the basic
scattering equation


For an ensemble of identical, randomly oriented
particles, the intensity of coherently, elastically
scattered radiation is dependant only upon the
magnitude of
q
, and can be expressed as:




N
= molecules/unit volume

V
= molecular volume


= contrast, the scattering density difference


between the scattering particle and solvent

P(q)

= form factor


particle shape

S(q)

= structure factor


inter
-
particle correlation distances



)
(
)
(
)
(
2
q
S
q
P
V
N
q
I



s






)
(
r
Inter
-
particle distance correlations
between charged molecules

D

D

D

D

D

-

-

-

-

-

D

-

D

-

D

….. gives a non
-
unity
S
(
q
) term

Sample requirements for
small
-
angle scattering
determination of
particle shape


Highly purified samples containing mono
-
disperse, identical particles without
significant inter
-
particle distance
correlations (
S
(
q
) = 1)


Use a final gel filtration step in the purification
immediately prior to measurement to
eliminate any aggregates


Us DLS to evaluate samples for potential
aggregates (mass fraction
aggregates<0.01%)

Essential preliminary

small
-
angle scattering
experiments


Explore the concentration dependence of the
small
-
angle X
-
ray scattering to determine if
S
(
q
)


1.


If
S
(
q
)


1, adjust the solution conditions by
changing pH, salt concentration, or decreasing
particle concentration to eliminate



Determine the particle mass, molecular volume,
and overall shapes of the components and their
complex (Guinier and
P
(
r
) analyses, shape
restoration)

Recall:
Guinier

Analysis


Guinier

showed that a plot (
lnI
(q)
vs

q
2)

gives a straight line of slope R
g
2
/3 and I(0)
intercept that can be interpreted in terms
of the concentration, contrast and volume
of the scattering particle.











3
)
0
(
ln
)
(
ln
2
2
g
R
q
I
q
I
2
)
(
)
0
(
V
N
I



Recall:
I
(
q
) and
P
(
r
) related by Fourier Transform


Fourier transform must be done using indirect methods
due to finite
q
-
range measured; quality samples and data
give well behaved transforms with certain characteristics

Sample

Protein
conc.
a

(mg/ml)


R
g

(Å)


D
max


(Å)

Porod
Volume
b


(10
3

x Å
3
)

Calculated
Volume
c


(10
3

x Å
3
)

MW
d

(kDa)

SSRL data

NL1
-
638
-

(A&B)

1.8

42.5
±

0.4

130

209
±

20

198

130/151/144

NL1/NX complex

3.6

46.8
±

0.2

155

*

275

181/201/199

University of Utah SAXSess instrument data

NL1
-
638
-

(A&B)

13.2

38.3
±

0.3

7.6

40.1
±

0.5

4.1

41.4
±

0.6

Inf. dilution

42.4
±

0.6

130

208
±

14

198

130/151/144

NL1
-
638

3.3

42.7
±

0.7

130

250
±

19

220

136/166/160

NL1
-
691

3.8

51.8
±

1.0

165

255
±

26

257

148/189/185

NL2
-
615

3.7

40.6
±

0.6

130

178
±

7

193

135/146/140

NL3
-
639

1.2

40.3
±

0.7

130

164
±

12

190

128/144/138

NL4
-
619

3.4

42.1
±

0.6

135

199
±

7

200

132/140/145

NL1/NX complex

19.7

40.9
±

0.3

15.7

40.8
±

0.2

9.8

44.0
±

0.3

8.7

43.7
±

0.4

6.6

44.5
±

0.4

4.5

45.2
±

0.5

3.8

47.7
±

0.9

155

*

Inf. dilution

47.7
±

0.8

155

*

275

181/201/199

NL1
-
638
-
Δ(A&B)

Complex

P(r) arbitrary units

Distance (Angstroms)

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

0

5

10

15

20

25

30

35

40

45

50

55

265,851

228,139
±

9,965

47.1
±

0.7

Complex

199,261

42.19
±

0.7

NL1
-
638
-
Δ(A&B)

Vol (Å
3
)

Calculated

Vol (Å
3
)

Experimental

Rg (Å)

Sample

184,172
±

7,778

Determining the size of your

scattering particle


Place data on an absolute scale (water
scattering) and use:




Orthaber et al. (2000)
J. Appl. Cryst. 33
, 218


Use a known mono
-
disperse protein
scatterer (such as lysozyme) and use:



Krigbaum and Kugler (1970)
Biochemistry 9
, 1216



Fischer et al. (2010) The molecular weight of proteins in
solution can be determined from a single SAXS
measurement on a relative scale.
J. Appl. Cryst. 43,
101


If you scale your data so that
I
(0) = 1, then:



where


In practice we can only calculate:


So Fischer et al calculated correction factors to relate the
‘apparent’ volume using
Q’

to the actual volume based on
1148 unique, known structures and their model profiles.


V

2

2
Q

Q

I
(
q
)
q
2
0


dq

Q
'

I
(
q
)
q
2

q
q
max
q
min

Planning the

neutron scattering experiment


Choose your data collection strategy
(solvent matching or contrast variation?)


Determine how much sample is needed


Decide which subunit to label


What deuteration level is needed in the
labeling subunit


See MULCh*

http://www.mmb/usyd.edu.au/NCVWeb/

*MULCh, Whitten et al, accepted
J. Appl. Cryst.

2007

MULCh


M
od
UL
es for the analysis of neutron
C
ontrast
variation data


Contrast,
computes neutron contrasts of the
components of a complex


R
g
,

analyses the contrast dependence of the radius of
gyration to yield information relating to the size and
disposition of the labelled and unlabeled components
in a complex



Compost
,
decomposes the contrast variation data into
composite scattering functions containing information
on the shape of the lab
\
led and unlabeled
components and their dispositions

Solvent matching


Best used when you are interested in the
shape of one component in a complex,
possibly how it changes upon ligand
binding or complex formation.


Requires enough of the component to be
solvent matched to complete a contrast
variation series to determine required
%D
2
O (~4 x 200
-
300

L, ~5 mg/ml).


Requires 200
-
300

L of the labeled
complex at 5
-
10mg/ml.

Solvent Match Point Determination

Apical view

Front view

Side view

90
°

90
°

Co
-
refinement of the


neurexin

positions and
orientations with respect
to NL1 give a model

against the X
-
ray and
neutron data
gives us a
model that we can map
autism
-
linked mutations

R451C

V403M

K378R

G99S

Comoletti, Grishaev, Whitten et al.

Structure 15
, 693
-
705, 2007.

Superposition of solution scattering and
crystal structure for NL
-
NX

Contrast variation


To determine the shapes and
dispositions of labeled and unlabelled
components in a complex


Requires


5 x 200
-
300

L (= 1


1.5mL) of your labeled complex at


5
mg/ml .


Deuteration level in labeled protein
depends upon its size.


Smaller components require higher levels
of deuteration to be distinguished.


Ideally would like to be able to take data
at the solvent match points for the labeled
and unlabeled components


Measure sample and solvent
blanks at each contrast point (use
a broad range of D
2
O
concentrations; e.g. 0,20,40, 80,
100% D
2
O)


Subtract solvent blank data from
sample


Sample to low
-
q

with sufficient
frequency to determine large
distances accurately (min. 15
-
20
points in the
Guinier

region)


Measure to high enough
q

to aid in
checking background subtraction
(
q

= 0.45
Å
-
1
)


q

= 0.01
-
.45 is typical range for 10
-
150
kDa

particles, usually requires
two detector positions

Effects of incoherent scattering
from
1
H on backgrounds


HCaM

measurement was
done in 42% D
2
O
to solvent match
the
HCaM
.
Objective was to
see
DCaM

in
presence of
HCaM
,
but without
interference
from
HCaM


Incoherent
scattering from
1
H
is a constant with q


X
-
ray scattering data from
LacI, with insert showing
Guinier plot with adequate
sampling.

Use
Rg

(from MULCh) for Sturhman analysis

2
2
2
2
)
(
D
f
f
D
H
D
H







2
2
2
2
2
)
(
)
(
D
f
f
R
R
f
f
H
D
D
H
D
H
D
H








2
2
2
2
D
f
f
R
f
R
f
R
D
H
D
D
H
H
m



2
2
2









m
obs
R
R
R
KinA

= 25.40
Å

R
Sda

= 25.3
Å

D = 27.0
Å

Use
Compost

(from
MULCh) to solve for
I(q)
11
,
I(q)
22
,
I(q)
12

I
1

I
12

I
2

)
(
)
(
)
(
)
(
12
2
1
22
2
2
11
2
1
q
I
q
I
q
I
q
I











Use SASREF7 to do rigid body
refinement of the components
against the scattering data (if you
have pdb files for components)



2

= 1.27


2

= 0.97


2

= 0.63


2

= 0.56


2

= 0.76


2

= 0.92


2

= 1.12


2

= 0.95


2

Incorporation of deuterium
up to 86%

of
the chemically Non
-
exchangeable protons
can be obtained by using D
2
O as the
deuterium source. Complete deuteration can
only be obtained by addition of
perdeuterated carbon source
(glucose or
glycerol).


Use mass spec to determine deuteration
levels.


The described protocols allow the deuteration content in recombinant proteins to be
predicted

Neutron scattering sample cells


Helma

quartz cells (high precision path
-
length,
suprasil
)


need lots of them!


Banjo
-
style (280

L per
1 mm path length) or
rectangular (170

L per 1 mm path length) cells
can be used


Path lengths are only good to 1%, so good idea
to measure sample and solvent background in
the same cell if practical, but experiment
logistics may prohibit that, so
often have to
‘fudge’ background subtractions


High incoherent scattering for
1
H means you
always want


1mm
1
H
2
O in the neutron beam to
avoid multiple scattering

Doing a Quality Experiment


After your final gel filtration step, check out your
samples with dynamic light scattering


Carefully calibrate your concentration assay


colorimetric assays are almost useless, extinction
coefficient is good if strong enough, quantitative
amino acid analysis can work


Compare your data to a well characterized standard(s)


For protein/DNA complexes, standards are more
difficult. Measure the partial specific volume of your
particle if you have enough sample


or use a good
model to calculate it, e.g. see MULCh or
http://geometry.molmovdb.org/NucProt/

Neutrons


Non
-
ionizing radiation


Penetrating


Wavelength and energies available that are suitable for
probing structures with dimensions 1
-
1000s
Å



Coherent scattering lengths that vary randomly with
atomic weight and large isotope effect for hydrogen


contrast variation


Large incoherent scattering cross
-
section for
1
H is a
source of noise in small
-
angle scattering


Interact weakly with matter and are difficult to produce
and detect


therefore should only be used when they
provide information that cannot be otherwise obtained.

Assessing the quality of

small
-
angle scattering results


Are there instrumental effects unaccounted for?


Are the scattering particles mono
-
disperse and identical or is there
a conformational ensemble?


Do you have dilute solution conditions?


Do the data show the expected Guinier and Porod behavior?


Is the
P
(
r
) “well
-
behaved?”


Are background subtractions accurate?


Have standards been measured?


How well characterized is the sample (purity, concentration)


Are errors appropriately handled


can you rely on

2
?

Jacques & Trewhella (2010)
“Small
-
angle Scattering for
Structural Biology;
Expanding the Frontier
While Avoiding the Pitfalls,”
Protein Science
19
, 642
-
657