Small-Angle X-ray Scattering - Learn the story of your protein Nozomi Ando @ Cornell - CHESS HP Bio Workshop 2021 - April 29 2021
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Extreme
Biophysics
RCN
Small-Angle X-ray Scattering
Learn the story of your protein
Nozomi Ando @ Cornell — CHESS HP Bio Workshop 2021 — April 29 2021
Structural biology techniques
Crystallography SAXS Cryo-EM
Cons: Cons: Cons:
• must crystallize protein • low resolution (10-20 Å) • hardware and software barriers
• crystallization artifacts • interpretation best done in reciprocal space • achieving < 3 Å resolution is not trivial
Pros: Pros: Pros:
• structure determination can be fast • insight into protein behavior in solution • game changer for large and slightly
• still the gold standard for atomic detail • connects structural data and tells a story heterogeneous samples
Tinoco, A. et al. ACS Catal 9: 1514–1524 (2019).
Davis, K. M. et al. PNAS 114: 10420–10425 (2017). Meisburger, S. P. et al. JACS 138, 6506–6516 (2016). Thomas, W. C. et al. Nature Commun 10, 2653 (2019).
Nozomi Ando — CHESS HP Bio Workshop 2021
Structural biology techniques
SAXS
SAXS fundamentals: How to
process and interpret data
Will Thomas
Pro tips on sample preparation:
How and more importantly, why?
Max Watkins
Cons:
• low resolution (10-20 Å) Advanced analysis: Extracting
• interpretation best done in reciprocal space more information from data
Pros:
• insight into protein behavior in solution Steve Meisburger
• connects structural data and tells a story
Meisburger, S. P. et al. JACS 138, 6506–6516 (2016).
Nozomi Ando — CHESS HP Bio Workshop 2021
A brief history of SAXS in structural biology
1400
1050
PubMed Results
André Guinier
(1968)
700
350
0
1970 1980 1990 2000 2010 2020
Year
Nozomi Ando — CHESS HP Bio Workshop 2021 Guinier, “30 Years of Small-Angle X-Ray Scattering”, Physics Today Nov. 1969
A brief history of SAXS in structural biology
1400
1050 1997: ab initio shape
PubMed Results
determination
700 1995: calculation of SAXS profiles
from atomic coordinates
350
0
1970 1980 1990 2000 2010 2020
Year
Svergun, Barberato, & Koch, J Appl Cryst. 28: 768–773 (1995).
Nozomi Ando — CHESS HP Bio Workshop 2021 Svergun, D. I. et al. J Appl Cryst. 30: 798–802 (1997).
A brief history of SAXS in structural biology
1400
A B C
1050 1997: ab initio shape
PubMed Results
90° 90°
determination
700 1995: calculation of SAXS profiles
from atomic coordinates
350
0
1970 1980 1990 2000 2010 2020
Year
Svergun, Barberato, & Koch, J Appl Cryst. 28: 768–773 (1995).
Nozomi Ando — CHESS HP Bio Workshop 2021 Svergun, D. I. et al. J Appl Cryst. 30: 798–802 (1997).
A brief history of SAXS in structural biology
1400
A B C
1050 1997: ab initio shape
PubMed Results
90° 90°
determination
700 1995: calculation of SAXS profiles
from atomic coordinates
350
0
1970 1980 1990 2000 2010 2020
Year
Svergun, Barberato, & Koch, J Appl Cryst. 28: 768–773 (1995).
Nozomi Ando — CHESS HP Bio Workshop 2021 Svergun, D. I. et al. J Appl Cryst. 30: 798–802 (1997).
Case study: Ribonucleotide reductase
NH 2 NH 2 NH 2 NH 2
N N N N
N N N N
A A A A
P P N N P P N ATP
N dATP
P P O
N N P P O
N N
O O
H H H H H H H H
H H H H H H H H
OH OH OH OH OH H OH H
NH 2 NH 2 NH 2 NH 2
GO STOP
N N N N
C C C C
N O N O N O N O
P P O P P O P P O P P O
H H H H H H H H
H H O H H O H H O H H O
OH OH OH OH OH H OH H
N ribonucleotide
N reductase (RNR) N N
NH NH NH NH
G G N
G N
G
N N
P P O
N P P
NH 2
O
N NH 2 P P O
N P P
NH
O
N NH
H H H H H H H H
H H H H H H H H
OH OH OH OH OH H OH H
O O O O
NH NH NH NH
U U U U
N O N O N O N O
P P O P P O P P O P P O
H H H H H H H H
H H H H H H H H
OH OH OH OH OH H OH H
Ribonucleotides Deoxyribonucleotides
Nozomi Ando — CHESS HP Bio Workshop 2021
Enzyme activity requires two proteins to transiently interact
E. coli Class Ia RNR
catalytic site
radical cofactor
(NDPs)
α2 subunit β2 subunit
Class I RNRs require inter-subunit radical transfer.
Nozomi Ando — CHESS HP Bio Workshop 2021
The hunt for the active complex
1961: Discovery 1990,1994: Crystal
of RNR in E. coli structures of subunits α2β2 docking model
proposed
Christina Zimanyi
1997: SAXS of α2 subunit 2009: Drug-inhibited
crystal structure
Nozomi Ando — CHESS HP Bio Workshop 2021SAXS reveals two complexes and connects structural data
0.15 μM
Negative-stain electron microscopy (EM)
physiological (1 - 2 μM)
1 - 10 μM
Analytical ultracentrifugation (AUC)
2 - 30 μM
Small-angle X-ray scattering (SAXS)
25 μM
X-ray crystallography
[RNR]
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.SAXS reveals two complexes and connects structural data
0.15 μM
Negative-stain electron microscopy (EM)
physiological (1 - 2 μM)
1 - 10 μM
full occupancy point
Analytical ultracentrifugation (AUC) 1 //
102 I(q) = f1 I175
1(q)μM
+ f2dATP
I2(q) αα44ββ44ring
0.8 ring
Fraction
101 0.6
I(q)
2 - 30 μM
0.4
100
Small-angle X-ray scattering (SAXS) 0.2 globular α24β42
compact
-1
10 0 //
0 0.04 0.08 0.12 0 10 20 30 40 50 170 180
q (1/Å) [dATP] (μM)
25 μM
X-ray crystallography
[RNR]
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.Structure of dATP-inhibited α4β4 complex
electron micrograph 1 asymmetric unit
Ed Brignole Christina Zimanyi
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.Structure of dATP-inhibited α4β4 complex
electron micrograph 2 asymmetric units
Ed Brignole Christina Zimanyi
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.Structure of dATP-inhibited α4β4 complex
α2
β2 β2
α2
3D EM reconstruction 2 asymmetric units
Ed Brignole Christina Zimanyi
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.Structure of dATP-inhibited α4β4 complex
α2
β2 β2
α2
3D EM reconstruction crystal structure
Ed Brignole Christina Zimanyi
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.SAXS connects conformational states and data from other techniques
allosteric regulation of activity
α2β2
α2 + β2 (active)
(inactive) α4β4 α8β8
(inactive) (inactive)
crystallization
kinetic stabilization
during radical transfer
Ando, Brignole, Zimanyi, Asturias, Drennan, Nocera, Stubbe, et al. (2011) PNAS, 108: 21046–21051.
Minnihan, Ando, Brignole, Asturias, Drennan, Nocera, Stubbe, et al. (2013) PNAS 110: 3835–3840.
Nozomi Ando — CHESS HP Bio Workshop 2021 Zimanyi, Ando, Brignole, Asturias, Stubbe & Drennan (2012) Structure 20: 1374–1383.Activity regulation in the enzyme family
E. coli human
P. aeruginosa
activity-regulating site
(ATP or dATP)
dATP binding to N-terminal ATP-cone domain promotes quaternary
structures that are incompatible with catalysis.
Ando, et al., Drennan. PNAS (2011). Zimanyi, et al., Drennan. Structure (2012).
Nozomi Ando — CHESS HP Bio Workshop 2021 Ando, et al., Drennan.. Biochem (2016). Johansson, et al. Structure (2016).Emergence of the class Ib RNRs
N
ATP-cone
E. coli class Ia RNR
Bacillus anthracis
(anthrax) Mycobacterium
tuberculosis
N
truncated sequence
Staphylococcus
Streptococcus
aureus
pneumonaie
class Ib RNRs
Class Ib RNRs lost allosteric activity regulation.
Nozomi Ando — CHESS HP Bio Workshop 2021Signatures of activity regulation…. in a class Ib RNR?
Mac Parker JoAnne Stubbe
ATP stimulates activity dATP inhibits
[ATP] (mM) [dATP] (µM)
The catch? This is a class Ib RNR.
Nozomi Ando — CHESS HP Bio Workshop 2021 Parker, Zhu, and Stubbe. Biochemistry (2014) 53: 766–776.protein_img
0 protein_img
0 protein_img
0 protein_img
100 0 protein_img
Anion exchange (AEX)-coupled SAXS
100 0
100
200 100
200 100
200
300 200
300 200
300
400 300
400 300
400
SAXS
500 400
500 400 detector
500
600 500
600 500 Will Thomas
600
700 600 sample
700 600
700
WAXS
800 700
800 700
800
detector
900 800
900 800 column
900
1000 900
1000 900
0
1000 200 400 600 800 1000
0
1000 200 400 600 800 1000
0
1000 200 400 600 800 1000
0 200 400 600 800 1000
0 200 400 600
scattering images
800 1000
elution
X-ray Steve Meisburger
anion-exchange chromatography-coupled SAXS
he use of this A C 0
SI Appendix,
nt cavity that 30 (2)
Holo -0.2Regals: regularized
[dAMP]
has been ob-
alternating least-squares
ln[I(q)/I(0)]
I(q) (a.u.)
f the class Ia 20 -0.4
Darren Xu
10 (1)
Apo -0.6
a Noncanonical 0 -0.8 Protein peaks:
cally observed 500
0.1
1000 0.05 -1 Background:
yed SAXS, a 1500 0 -1 0 0.4 0.8 1.2 1.6 2
voluting mix- Frame Number q (Å )
q2 (Å-2) x10-3
o-NrdE were B D Meisburger,
// Taylor, Khan, Zhang, Fitzpatrick, and Ando. JACS (2016) 138: 6506 - 6516.
Appendix, Fig. Parker, Maggiolo*, Thomas*, Kim, Meisburger, Ando†, Boal†, and Stubbe†. PNAS (2018) 55: 201800356–10.
to Nozomi
an in-line 104 HP(3)
Ando — CHESS Bio Workshop 2021 42 Meisburger*, Xu*, and Ando IUCrJ (2020) 8: 225-237.
y path with a 38
Rg (Å)
.6. Scattering
103 (2)
of the elution 34
esponding to
30
to those seen
) (a.u.)
102
ppendix, Fig. (1)
26
//AEX-SAXS reveals a new oligomeric form
specificity site dAMP site
(S-site) (I-site)
PDB: 6CGL
S
I
expected “S-dimer” instead discovered “I-dimer”
Endogenous ligand (dAMP) binds to truncated N-terminal domain.
Meisburger, Taylor, Khan, Zhang, Fitzpatrick, and Ando. JACS (2016) 138: 6506 - 6516.
Parker, Maggiolo*, Thomas*, Kim, Meisburger, Ando†, Boal†, and Stubbe†. PNAS (2018) 55: 201800356–10.
Nozomi Ando — CHESS HP Bio Workshop 2021 Meisburger*, Xu*, and Ando IUCrJ (2020) 8: 225-237.Mapping allosteric transitions with SAXS
Step 1: Anion-exchange (AEX-SAXS)
nd further supports the use of this A +C Regals
- and triphosphates (SI Appendix, 0 Will Thomas
tes a second adjacent cavity that
e simultaneously, as has been ob-
30 Holo -0.2 [dAMP] identify liganded states
ln[I(q)/I(0)]
I(q) (a.u.)
two cone domains of the class Ia 20 -0.4
10 Apo -0.6
-NrdE Is Predominantly a Noncanonical 0 -0.8
e if the crystallographically observed 500
1000 0.1
solution, we employed SAXS, a
well suited for deconvoluting mix-
1500
Frame Number
0
0.05
-1
q (Å )
-1
0 0.4 0.8 1.2 1.6 2
x10-3
Step 2: Titration SAXS +
q2 (Å-2)
Because apo- and holo-NrdE were B D // singular value decomposition (SVD)
chromatography (SI Appendix, Fig.
(3)
NrdE was loaded onto an in-line 10 4 42
directly into the X-ray path with a
Parker, et al. PNAS 38
Rg (Å)
0-mM NaCl at pH 7.6. Scattering (2018) 55: 201800356–10.
103 (2)
ously over the course of the elution
two major peaks corresponding to
34
= x x
30
ons (Fig. 7A), similar to those seen
I(q) (a.u.)
escribed earlier (SI Appendix, Fig.
102
Step
(1) 3: Size-exclusion
26 (SEC-SAXS) // +
et was then mathematically decom- 0 2 4 6 8 10 300
of four distinct scattering compo- 10 1 evolving factor analysis
[dAMP] (µM) (EFA)
profile and SAXS curve, using a E 0
quares (ALS) method developed in-
-0.4 0 µM dATP
. Mathematical decomposition by 10 0
ed in the context of size-exclusion
ln[I(q)/I(0)]
-0.8
S, where the sequential nature of 20 µM dATP
ard means to identify where indi- 10-1 -1.2 identify structural
34). In the context of AEX–SAXS,
transitions
20 -1.6 50 µM dATP
tering varies in a complex manner
res.
0
due to the NaCl gradient, compli- -20 -2
10-2 10-1 0 0.2 0.4 0.6 0.8 1
e the AEX–SAXS data, we added q (Å-1) q2 (Å-2) x10-3
he ALS method, so that background
(by selection of a large Lagrange
cally during elution. With this ap-
Fig. 7. SAXS reveals the effects of adenine nucleotides on NrdE oligomeri-
zation. (A) AEX–SAXS dataset of aiNrdE. (B) SAXS profiles of the two protein
structural modeling
Meisburger, et al. (2016) JACS, 138: 6506–6516..
components from AEX–SAXS are shown offset with fits and corresponding
taset could be represented by two
residuals (res.) shown below with the same coloring. Set 1: The monomer
two partly overlapping peaks (SI
component (gray) is well described by the calculated profile of a NrdE
subtraction of the variable scatter- monomer (overlaid blue curve). Sets 2 and 3: The dimer component (gray) is
been reported
Nozomi for ion
Ando — exchange-
CHESS HP
wellBio Workshop
described 2021profile of the noncanonical dimer (overlaid Thomas,
by the calculated Brooks, Burnim, Bacik, Stubbe, Kaelber, Chen, and Ando. Nature Comm (2019) 10: 2653.
nce measurement (35), the regular- orange curve), whereas that of a canonical dimer (overlaid magenta curve)
AXS data we present here represents shows a worse fit. (C) Titration of 0- to 300-μM dAMP into 2-μM apo-NrdE
ackground separation. leads to a steeper slope in the Guinier region of the SAXS profiles (solid lines,
X–SAXS data by regularized ALS blue to red) with the AEX-derived monomer (blue dashed line) and dimer (red
n of two protein components (SI dashed line) components shown for reference. (D) As [dAMP] increases, the Rg
increases from that of a monomer and approaches that of the AEX-derived
ution of apo-NrdE corresponds to
dimer (red dashed line), saturating at stoichiometric concentrations. (E) The
hich was found to have a radius of addition of dATP to 1-μM holo-NrdE leads to the formation of increasingly
(Guinier Rg = 27.8 ± 0.1 Å) and a large structures, as indicated by an increasingly steep Guinier region of the
ay curve in set 1) that is well de- SAXS profile. A transformation of this plot further shows that the species
tering from a monomer model of becomes increasingly extended with [dATP] (SI Appendix, Fig. S11).Discovery of a new inhibition mechanism
monomer
monomer
S-dimer
I-dimer
filament
I-dimer
I-dimer
From SAXS, we knew:
Endogenous ligand binds 1 site per
monomer to produce an I-dimer.
cryo-EM
dATP converts both the monomer structure
and I-dimer to the same filament.
∴ There are 2 binding sites for dATP
& the I-dimer is part of the filament.
“packing peanut on a post-it” model
Thomas, Brooks, Burnim, Bacik, Stubbe, Kaelber, Chen, and Ando. Nature Comm (2019) 10: 2653.2.5 Å crystal structures
4.8 Å cryo-EM structure
4.7 Å cryo-EM structure
SAXS model
Nozomi Ando — CHESS HP Bio Workshop 2021 Thomas, Brooks, Burnim, Bacik, Stubbe, Kaelber, Chen, and Ando. Nature Comm (2019) 10: 2653.a b c
An elegant form of convergent allostery
a 2
b 2
1
2
NrdEF EM
0.8 AsymmetricResidues
Symmetric
b c 45-50
10 0 Apo-NrdE 2
Ixq2
monomer 0.6
dE
*
Holo-NrdE
Asymmetric tetramer 2
I(q)/I0
I-dimer
Apo-NrdE
* 0.4
I-dimer 10 -2
Holo-NrdE * 2
+ dATP
2
Monomer 0.2
10 -4
10 -2
500 10
Å-1 500 Å
q (Å-1) q (Å-1) 0.05 0.1 0.15 0.2
2
e q (Å )-1
Symmetric tetramer
α2
2
α2
dATP
full range of
~50 Å
ATP motion allowed
β2
S-dimer
NrdF
Symmetric Asymmetric
I-dimer β22 C-terminus
Docking model Docking model
2 2 2 2
dATP at I-site sequesters ɑ2β2 units into a filament.
ATP at I-site acts to siphon the active form of ɑ2β2.
Nozomi Ando — CHESS HP Bio Workshop 2021 Thomas, Brooks, Burnim, Bacik, Stubbe, Kaelber, Chen, and Ando. Nature Comm (2019) 10: 2653.Take-home messages
Low-resolution but provides incredible insight into
SAXS protein behavior in solution.
Easy to change solution conditions and do a
comprehensive search of conformational space.
SAXS connects data from other techniques.
SAXS allows comparative study of protein homologs.
SAXS tells a story.
Nozomi Ando — CHESS HP Bio Workshop 2021You can also read
