Imaging RNA structures and folding intermediates using electron cryo-microscopy
Tobin R. SosnickDept. of Biochemistry and Molecular Biology
Institute for Biophysical DynamicsUniversity of Chicago
RNA can form irregular tertiary structures
Sarcin Ricin loop
tRNA
P RNA subdomain
Group I Intron
Ribosome
How do they obtain these structures?Kinetics --- Thermodynamics --- Structure
Only the sequence is needed!
Primary Sequence
(nucleotides amino acids)
Native
RNA
Unfolded
“Mother Folding”
Non-specific binding occurs at low [Me], stabilizes secondary structures Specific binding at higher [Me2+] is involved in tertiary folding transitions.
duplex tRNA P4-P6 domain
2. High occupancy metal sites are observed in crystal structures of tertiary RNAs (often Me2+).
Me2+
“Mg2+ core”
Two classes of cation-RNA interactions1. non-specific binding (counter-ion condensation, Me+ or Me2+ )
+ +
+
+
+
+
+
+
+
+
+
+
+
++++ +
+
+
+
++
+
+
+
+
+
+
+
+
+
++
+
+
+
Metal requirement: Tertiary RNAs typically require divalent cations to fold.
P15.1
G U U C U U A A C GU U C G GGUAAUC
G
A
A A A C C CA A A U
UUU
GGUAGGG
GAAC CU U C U U
AA C G GA
AUUC
AACGG
AGAGAAGG
A C AGA A UG
CUUUCUGU
AGAUAGAUGAUUGCC G C
CUG
A GUA
CG
A GG
UG A U
GAG
CC G
UUU
GCAG
UA
CGA
UGG A A C A
AAACAUGGCUU
ACAGAACGUUAGA
CCACUU
GU
CC
UGCUCGC
ACGG
UGC
UG
AGAUGCCCGUAG
UG U UCGUGC
CU
AG
CGA
AGUC
AUA
A GCUAGG
GCAGUCUUUA
G A GG C U GAC G
GCAGGA
AAAAAGCC
UAC
GUCUUC GG A U A
U G GCU G A G
UAU C C U
U G AAA GU
GCCACA
GU G A C G A A
GU CU C AC U A
GAA
AUGGUGAGAGUGG
AA
CGCGGUAA
ACCCC U C GA G C GA G
CUGCAGAUCUUG A A U C U GU A G
AGGAAA
1
20
40
60
100
120
140
160
200
220
260
280
300
320340
360
380
240
400
80
P1P2
P4
P15
P18
P19
P3
P5
P7
P5.1
P8
P9P10
P11
P10.1P12
Bacillus stearothermophilus Rnase P RNA
Pace and coworkers, PNAS 2005
Catalytic C-domain (~255 nt)
Specificity S-domain (~150 nt)
3.0
4.0
5.0
6.0
fluo
r.
C
D(2
87)
CD
(260
)
1.3
1.4
1.5
1.6
1.7
2.2
2.3
2.4
2.5
Abs (260)
0 . 0 1 0 .1 1
0 .45
0 .50
0 .55
0 .60
M g 2 + ( m M )
Mg2+-induced equilibrium folding of C-domain of P RNA
UIMgK
U I
IeqUi
U2
U1
|
UIMgK
At low [Me2+], non-specific (and specific) binding,
NINMgK
At high [Me2+], energetics of dominated by specific
binding
INMgK
N
Fang, Pan & SosnickBiochemistry, 1999 Mg2+
+n
Limited by Me2+ binding site formation: Defines tertiary structure
Fast, local conformational search
Limiting stepConsolidation of specific
metal binding site
I2k
98%buried
Rg = 38 Å
N100%buried
Rg = 180 Å
0% surface buried
Uurea
≤ 1msec collapse, 2o +some 3o,non-specific and some specific Mg2+
85%buried
Rg = 46 Å
Ieq fast
Rg = 39 Å
I1k
98 %buried
fast
Summary of C-domain folding
Fang, Pan & Sosnick, PNAS, 2002
Structural characterization of intermediates
U I N
?Mg2+ Mg2+
B. subtilis Specificity domain of RNase P RNA
Krasilnikov et al, Nature 2003
KEV1 T1 DEPCV1 DMS
C
CU
P9P8
J12/11
220
100
UG
•
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
••
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
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•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
•
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
••
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
•
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
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AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
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•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
••
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
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•
•
•
AA
A
U
U
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GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
•
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
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•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
••
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
•
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
••
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
•
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
C
CU
P9P8
J12/11
220
100
UG
••
•
A U AA G C U A GGC
C C C G GG G G
A
A A U
U
U U G G
GG
C C
CCU
UU
A
A A
A
•••• •
a GG
Gc CC
C
U
g a GG
Gc CC
C
U
g
A
AA
GG
GGG CC
CC U
U G•
AAAAA
G
GG
G
G
G
C
C
C
U
U
U
U
U
U
U
CC
A
A
A
A
A
G
G
•
••
•
•
•
AA
A
U
U
U
GG
G
•
CC
A
A
AA
A
U
U
U
GG
G
•
CC
A
A
A
AA
AA
G
GGG
G
C
C
C
U
U
U
UU
A
AG
•
AG
A
A
A
A
G
GC
C
A
G
GG
C
C
U
G
A
C
3’
5’
P10.1
P12
P7 J11/12
P11
P10
140200
86
239
180
160
120
Site-resolved informationfrom chemical and nuclease cleavage
U to I transition
Four-way junction
J11/12 module
I to N transition
Core
TL-receptor
native
Ieq contains J11/12 module & four-way junction
0 40 80 120
0.000
0.005
0.010
0.015
0.020
Rg ~ 30 Å
Experimental SAXS Crystal Structure
P(r
)
r (Å)
P(r) – general shape, from small-angle X-ray scattering
Rg – overall sizeAPS, BioCat beamline
N
Ieq
Rotate both P10.1 and P12 arm
∆Rg ~ 8 Å
Disrupt TL-receptor interaction
∆Rg ~ 2 Å
Rotate P10.1 further
∆Rg ~ 5 Å
With Eric WesthofBaird et al. JMB 2005
Start from the crystal structure…Modeling the intermediate with experimental constraints
(SAXS, Nuclease and chemical mapping)
Ieq N
Intermediate Structure
Role of metal ions in folding cooperativity
Direct mediation of long-range contacts
Indirect mediation –metal binding coupled to large-scale conformational change
U IeqN
S-domain
NIeq
Intermediate Structure (model)
Large molecules light up on EM
• Ribosome – 3 MD
• Proteasome – 750 kD
• Generally accepted lower limit ~200 kD
Glaeser group, UC Berkeley,data from Frank lab, Wadsworth Center
Hu et al, Mol. Microbiology, 2006
But nothing beats a real picture…
Catalytic domain RNase P RNA
Direct imaging of ‘small’ RNAsW. Chiu & S. Ludtke, Baylor
82 kDSide
Top
Front
Start with molecules with known crystal structures to assess feasibility
(blind reconstructions)
Ieq reconstruction
When the intermediate is stably populated it can be
directly imaged!
Native S-domain
I∆25 comparison with reconstruction
Model vrs SAXS & CryEM reconstructions
ModelCryoEM
SAXS
Add an extension to enhance image
Native
Ieq with P9ext
0.01 0.1 1 10
4.2
4.5
4.8
5.1
[MgCl2] (mM)
CD
260
2.6
2.8
3.0
3.2
3.4
CD
278
Folding behavior unchanged
Top view
Front view
Ieq with P9ext
Ieq dimensions: no salt dependence below 1 M NaCl
Why doesn’t flexibility blur the image?
0.01 0.1 1
32
34
36
38
40
42
Rg
(A)
[NaCl] M
Mg2+-native
P10.1
P9
P10.1
P9
P10.1
P9
P10.1
P9
Not just electrostatics holding Ieq in an extended state: Defined thermodynamic well
Structure in the core? All-atomsimulations
RNA structure determination using CryoEM + modeling + all-atom simulations:
A rapid alternative to crystallography?
CGGAUAGGC
UUCUGCAUCCG
U UGAGUAUA
AAAAAGGACG
CCUUGAA
AGUG
CCACAG
UGACGAAG
GCAGUCGGAGAUUUCUGACGG
GA
CCCAUG
GCGCAAG
GUGAU UC CA UC AGA
AAUGGUGAG
UC
GAAUACUGA
AGCGA
UCC
GCGACUUG
UGAU
230
220
210
200
140 130
120 110
100
150
160
170
180
190
90P5
P7
P8
P9
P10P10.1
P11 P12..
.
. .
modelingAll-atom
simulations
Thoughts:
Include “folding” to help restrict the conformational search in prediction
What about folding cooperativity?
Acknowledgments• Tao Pan
– Nathan Baird – Haipeng Gong– Shahnawaz Zaheer
• Wah Chiu, Steve Ludtke (Baylor, CryoEM) • Eric Westhof (Strasbourg, modeling)• Karl Freed (Univ. of Chicago, simulations)
NIH
