Transcript ppt - Erice Crystallography 2006 IT Support

Single-particle reconstruction in the
absence of symmetry
Absence of symmetry means
much larger data collection
Absence of crystal packing means
larger degree of variability, i.e.,
heterogeneity of particle set
Low-resolution density maps must
be interpreted in terms of atomic
structures
High-resolution Single-Particle Cryo-EM:
Low (nonexisting) symmetry
• Bridget Carragher: Case studies in automation
• Jose-Maria Carazo: Dealing with conformational
variability by advanced classification and alignment
methods
• Joachim Frank: (Quasi-) atomic models of the
ribosome in different functional states – flexible fitting
(Quasi-) Atomic Models of the Ribosome in Different
Functional States, by Cryo-EM and Flexible Fitting
Joachim Frank
Howard Hughes Medical Institute, Health Research, Inc., Wadsworth
Center, Albany, New York
Department of Biomedical Sciences, State University of New York at
Albany
Supported by HHMI, NIH R01 GM55440, NIH R37 GM29169, and the National Center for
Research Resources (NCRR/NIH)
Elongation of the Polypeptide Chain by One Amino Acid
aa-tRNA
>>
accommodation
peptidyl transfer >>
translocation >>
aa-tRNA
>>
accommodation
The Role of the Elongation Factors EF-G and EF-Tu
translocation
tRNA selection
110,000
projections
7.8 Ǻ
Spahn et al. (2003) in prep.
73,000
projections
11.5 Ǻ
Gabashvili et al. (2000) Cell
Wheat germ
52,000 projections
9.5 Å
Halic et al., NSMB 2005
The 70S Ribosome, Seen by Two Modalities of
Imaging
X-ray structure (T. thermophilus)
(Yusupov et al., Science 2001)
Cryo-EM map (E. coli)
(Gabashvili et al., 2000)
The elongation cycle as seen by cryo-EM using antibiotic
and GTP nonhydrolyzable analogs (no X-ray studies thus far)
peptidyl transfer
decoding
translocation
kirromycin
GDPNP
fusidic acid
thiostrepton
GDPNP
Functional dynamics of the ribosome
In each binding event observed so far, both ribosome and the
ligand (e.g., the elongation factor) undergo conformational
changes.
“Induced fit” phenomenon
Example: the “Ratchet Motion”
-EF-G
+ EF-G
•GDPNP
1) When EF-G binds to the ribosome, the small subunit rotates counter-clockwise
relative to the large subunit. (Frank and Agrawal, Nature 2000)
2) EF-G is no longer in X-ray GDP conformation (Agrawal et al., PNAS 1998)
What is the Purpose of the Ratchet Motion?
Mechanism of mRNA Translocation, in Two Phases
PHASE I:
mRNA moves
along with 30S,
relative to 50S
(lock is closed)
PHASE II:
30S moves
back, relative
to mRNA and 50S
(lock is open)
Flexible Fitting
• We wish to explain the conformational changes observed in the
low-res density map in terms of changes in the atomic structure.
We achieve this by “molding” the X-ray structure of the static
ribosome into the density map, by a process of flexible fitting.
The following two computational methods can be used:
• Normal Mode Analysis-guided flexible fitting (NMFF): molecule
is modeled as an elastic network (“balls connected with
springs”); only small amplitudes allowed.
• Real-space refinement: provides multi-fragment docking,
preserving structural integrity as much as possible.
The resulting quasi-atomic models have enormous heuristic value,
allowing dynamic changes of the system to be followed, and
testable hypotheses to be formulated.
Normal Mode Analysis Applied to X-ray Structures
The preferred modes of motion
are implicit in the gross
molecular architecture.
For example, the “ratchet” motion triggered
by the binding of EF-G is predicted by
normal mode analysis:
•Relative Rotation of Small Subunit
•L1 stalk pivoting
Normal-mode Analysis guided fitting
deforms structure along its normal modes
such that optimal agreement is reached
with the density map.
Animation
Tama et al., PNAS
Tama et al., PNAS 100 (2003) 9319
Jernigan, J. Struct. Biol. (2004)
Wriggers: NMA of low-res. density maps.
Fitting via Real-Space Refinement (Chapman, 1995)
Rgeom = stereochemical term
Real-space Refinement
geometry restraint
density restraint
Rρ
Rgeom
energy minimization, TNT, CNS
Dynamic events we have analyzed by
real-space refinement
•
•
•
Translocation: EF-G-induced ratchet motion, motion of a factor-binding
component of the ribosome called “GTPase-associated center” (GAC), and
motion of L1 stalk
Decoding/tRNA selection: motion of GAC and kinking/distortion of the tRNA
Signal peptide (SecM)-induced translational arrest: for the ribosome to allow
lateral insertion of membrane-intrinsic protein in co-translational protein
translocation
Each analysis consists of a comparison of two maps via RSR. PDB-formatted
coordinates can then be displayed using any molecular graphics package.
Very effective and informative display modes:
1) animation – rotate Ribbons representations while alternating between of the
two versions of the structure.
2) color the Ribbons representation of one structure according to the magnitude
of the RMSD between the two structures .
3) color the secondary structure diagram of one structure according to the
magnitude of the RMSD between the two structures
Steps to Follow in Real-Space Refinement
1) Decide on a division into stable fragments. Here are the choices for the
ratchet motion:
16S RNA
43 pieces
23S RNA
62 pieces
5S RNA
4 pieces
Proteins: most retained as single rigid units.
exceptions: S2, S7, S13; L2, L3, L5, L9, L11, L18, L24,
which were cut into major domains.
Total number of rigid pieces: 162. Is this overfitting? No:
Number of degrees of freedom: (100Ǻ/10Ǻ)3 *4/3π ~ 4000
2) Use manual or automated rigid-body docking for pre-alignment
3) Use RSRef program
Gao et al., Cell 113 (2003) 789-801
E. coli rRNA
16S
23S
5S
Real Space Refinement Using RSRef:
Ratchet motion
Map resolution
Initial CCC
Final CCC
Initial R-factor
Final R-factor
Initial vdW close
Final vdW close
Initiation-like
11.5 Å
0.53
0.71
0.29
0.23
>10,000
~1,900
Gao et al., Cell 113 (2003) 789-801
EF-G bound
12.3 Å
0.37
0.67
0.32
0.24
>10,000
~1,200
Gao et al., Cell 113 (2003) 789-801
Gao et al., Cell 113 (2003) 789-801
RSREF applied to EF-G-triggered ratchet-like rotation
Color mapping shows where changes occur maximallly.
Dynamics of tRNA Selection and Accommodation: Cryo-EM
Snapshots in Three States
unbound
“A/T”
post-translocation
Phe-tRNAPhe•EF-Tu•GDP•kir
ready for next tRNA
tRNA selection
“A/A”
accommodated
tRNA “approved”
Valle et al., NSMB 10 (2003) 899
Real-Space Refinement Using RSRef: binding
of ternary complex (A/T state)
Unbound
Map resolution
11.5 Å
Initial CCC
0.53
Final CCC
0.71
Initial R-factor
0.29
Final R-factor
0.23
Initial vdW close >10,000
Final vdW close
~1,900
Sengupta et al., in preparation
A/T state
12.5 Å
0.48
0.67
0.36
0.26
>10,000
~4,000
H43/H44
(GAC)
Sengupta et al., in preparation
RSREF applied to A/T state (ternary complex bound) and unbound state:
GAC moves strongly
GAC (L11+helices 42, 43, 44 of 23S rRNA) movements in response to
(1) GTP hydrolysis (open  half-closed) and (2) binding of ternary
complex (half-closed  closed)
“closed”
“half-closed”
“open”
KT-42
Frank et al., FEBS Lett. 2004
translocon
lateral insertion into lipid
Co-translational insertion of transmembrane protein, signaled by
SecM signal sequence that is in transit in the tunnel, requires
translational arrest
K. Mitra
HHMI
Wadsworth Center
SecM-induced conformational changes in the ribosome
analyzed by RSRef (translational arrest)
K. Mitra et al., Mol. Cell 2006
Presence of SecM is probably sensed by L4 and L22 “fingers”, producing conformational signal.
Conformational changes, and their putative roles in translational arrest
Mitra et al., Mol. Cell 2006
Control of dynamic study with RSRef: use identical
samples; re-do all steps of sample prep, EM, image
processing and RSRef
Mitra et al., Mol. Cell 2006
Result: RMSD between pairs of coordinates of same residue is below
2 Å everywhere, (see Rossmann’s rule of thumb: ratio 1 to 5)
Overview over docking and fitting procedures
(Fabiola and Chapman, 2005)
• Global rigid body search for initial configuration
SITUS (Wriggers and Chacon, 2001) use of “code vectors”
COAN (Volkman et al., 2003) 6-D exhaustive search; consider solution set
DOCKEM (Roseman, 2000) 6-D exhaustive search; local normalization of cross-correlation
• Final refinement
URO (Navaza et al., 2002)refinement in reciprocal space
NMFF-EM (Tama et al., 2004) normal-mode analysis guided fitting
RSREF (Chapman et al., 1995; 2005) real-space refinement
• In between
EMFIT (Rossmann, 2000) variety of target functions; refinement in reciprocal space
SITUS (Wriggers and Chacon, 2001)
CHARMM coarse-grained search combined with Monte-Carlo optimization (Wu et al.,
2003)
Conclusions
• Real-space refinement can be used to construct
quasi-atomic models depicting snapshots of a
dynamic process.
• Such models have great heuristic value as they
allow local conformational changes underlying
global motions to be followed.
• The fit of the ribosome with a number of pieces in
the order of ~150 represents a conservative use of
RSRef
• Insights have been obtained for translocation,
tRNA selection, and SecM-induced translational
arrest.
Contributors
Members of the group:
Haixiao Gao
Bob Grassucci
Kakoli Mitra
Mikel Valle – now at CNB, Madrid
Jayati Sengupta
Christian Spahn – now at Charite, Humboldt University, Berlin
Collaborators within:
Patrick Van Roey -- Wadsworth
Rajendra Agrawal -- Wadsworth
Outside collaborators
Andrei Sali and Narayanan Eswar, UCSF
Måns Ehrenberg and Andrej Zavialov, Uppsala University
Michael Chapman, Felcy Fabiola, and Andrej Korostelev, Florida State
Steve Harvey and Scott Stagg, Georgia Tech
Charles Brook and Florence Tama, Scripps