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 Chainby 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. 11.5 Ǻ 73,000 projections 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) Cryo-EM map (E. coli) (Yusupov et al., Science 2001) (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 translocation decoding fusidic acid thiostrepton GDPNP kirromycin 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-SpaceRefinement (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 Initiation-likeEF-G bound Map resolution 11.5 Å 12.3 Å Initial CCC 0.53 0.37 Final CCC 0.710.67 Initial R-factor 0.29 0.32 Final R-factor 0.230.24 Initial vdW close >10,000 >10,000 Final vdW close ~1,900 ~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” “A/A” post-translocation Phe-tRNAPhe•EF-Tu•GDP•kir accommodated ready for next tRNA tRNA selection tRNA “approved” Valle et al., NSMB 10 (2003) 899
Real-Space Refinement Using RSRef: binding of ternary complex (A/T state) Unbound A/T state Map resolution 11.5 Å 12.5 Å Initial CCC 0.53 0.48 Final CCC 0.710.67 Initial R-factor 0.29 0.36 Final R-factor 0.230.26 Initial vdW close >10,000 >10,000 Final vdW close ~1,900 ~4,000 Sengupta et al., in preparation
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