Single-particle reconstruction in the absence of symmetry

Absence of symmetry meansmuch larger data collection

Absence of crystal packing meanslarger degree of variability, i.e.,heterogeneity of particle set

Low-resolution density maps mustbe 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 ChainElongation of the Polypeptide Chain by One Amino Acidby One Amino Acid

peptidyl transfer >> translocation >>aa-tRNA >>accommodation

aa-tRNA >>accommodation

tRNA selection

The Role of the Elongation Factors EF-G and EF-Tu

translocation

Gabashvili et al. (2000) Cell

Spahn et al. (2003) in prep.

7.8 Ǻ

11.5 Ǻ73,000projections

110,000projections

Halic et al., NSMB 2005

52,000 projections 9.5 Å

Wheat germ

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)

translocationdecoding

kirromycinGDPNP

fusidic acidthiostreptonGDPNP

peptidyl transfer

The elongation cycle as seen by cryo-EM using antibioticand GTP nonhydrolyzable analogs (no X-ray studies thus far)

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

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)

Example: the “Ratchet Motion”Example: the “Ratchet Motion”

-EF-G

+ EF-G •GDPNP

What is the Purpose of the Ratchet Motion? What is the Purpose of the Ratchet Motion? Mechanism of mRNA Translocation, in Two PhasesMechanism of mRNA Translocation, in Two Phases

PHASE I:mRNA movesalong with 30S,relative to 50S(lock is closed)

PHASE II:30S movesback, relativeto 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 StructuresNormal Mode Analysis Applied to X-ray Structures

The preferred modes of motion are implicit in the gross molecular architecture.

For example, the “ratchet” motion triggeredby 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 modessuch that optimal agreement is reached with the density map.

Tama et al., PNAS Animation

Tama et al., PNAS 100 (2003) 9319Jernigan, J. Struct. Biol. (2004)Wriggers: NMA of low-res. density maps.

Fitting via Real-Space Refinement (Chapman, 1995)

Rgeom = stereochemical term

Rρ

Rgeom

geometry restraint density restraint

energy minimization, TNT, CNS

Real-space Refinement

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 pieces23S RNA 62 pieces 5S RNA 4 piecesProteins: 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

23S

16S

5S

E. coli rRNA

Real Space Refinement Using RSRef:Ratchet motion

Initiation-like EF-G boundMap resolution 11.5 Å 12.3 ÅInitial CCC 0.53 0.37Final CCC 0.71 0.67Initial R-factor 0.29 0.32Final R-factor 0.23 0.24Initial vdW close >10,000 >10,000Final 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 rotationColor mapping shows where changes occur maximallly.

Gao et al., Cell 113 (2003) 789-801

Dynamics of tRNA Selection and Accommodation: Cryo-Dynamics of tRNA Selection and Accommodation: Cryo-EM Snapshots in Three StatesEM 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 stateMap resolution 11.5 Å 12.5 ÅInitial CCC 0.53 0.48Final CCC 0.71 0.67Initial R-factor 0.29 0.36Final R-factor 0.23 0.26Initial vdW close >10,000 >10,000Final vdW close ~1,900 ~4,000

Sengupta et al., in preparation

RSREF applied to A/T state (ternary complex bound) and unbound state: GAC moves strongly

H43/H44 (GAC)

Sengupta et al., in preparation

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”

Frank et al., FEBS Lett. 2004

KT-42

K. MitraHHMIWadsworth Center

Co-translational insertion of transmembrane protein, signaled by SecM signal sequence that is in transit in the tunnel, requires translational arrest

translocon lateral insertion into lipid

SecM-induced conformational changes in the ribosome analyzed by RSRef (translational arrest)

Presence of SecM is probably sensed by L4 and L22 “fingers”, producing conformational signal.

K. Mitra et al., Mol. Cell 2006

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

Result: RMSD between pairs of coordinates of same residue is below 2 Å everywhere, (see Rossmann’s rule of thumb: ratio 1 to 5)

Mitra et al., Mol. Cell 2006

Overview over docking and fitting procedures (Fabiola and Chapman, 2005)

• Global rigid body search for initial configurationSITUS (Wriggers and Chacon, 2001) use of “code vectors”COAN (Volkman et al., 2003) 6-D exhaustive search; consider solution setDOCKEM (Roseman, 2000) 6-D exhaustive search; local normalization of cross-correlation

• Final refinementURO (Navaza et al., 2002)refinement in reciprocal spaceNMFF-EM (Tama et al., 2004) normal-mode analysis guided fittingRSREF (Chapman et al., 1995; 2005) real-space refinement

• In betweenEMFIT (Rossmann, 2000) variety of target functions; refinement in reciprocal spaceSITUS (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 GaoBob GrassucciKakoli MitraMikel Valle – now at CNB, MadridJayati SenguptaChristian Spahn – now at Charite, Humboldt University, Berlin

Collaborators within:Patrick Van Roey -- WadsworthRajendra Agrawal -- Wadsworth

Outside collaboratorsAndrei Sali and Narayanan Eswar, UCSFMåns Ehrenberg and Andrej Zavialov, Uppsala UniversityMichael Chapman, Felcy Fabiola, and Andrej Korostelev, Florida StateSteve Harvey and Scott Stagg, Georgia Tech Charles Brook and Florence Tama, Scripps