Structural prediction combined with MDFF

Yassin et al. have recently published a work in PLoS One combining systematic structural prediction with MDFF in order to model the mitochondrial initiation factor 2 (IF2), which allowed the authors to test structural hypotheses regarding a mitochondrial-specific sequence insert.

Computational exploration of structural hypotheses for an additional sequence in a mammalian mitochondrial protein.
Aymen S. Yassin, Rajendra K. Agrawal, and Nilesh K. Banavali. PLoS One, 6, e21771, 2011.

Background:
Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.

Results:
The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2mt predictions differ from the observed IF2mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.

Conclusions:
By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2mt cryo-EM map to get reasonable quasi-atomic IF2mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.

Structure of a no-go mRNA decay complex bound to a stalled 80S ribosome

A recent work by the Beckmann group (University of Munich, Germany) published in Nature Structural and Molecular Biology describes a sub-nanometer cryo-EM reconstruction of a no-go mRNA decay complex bound to a stalled 80S ribosome. Homology models were manually docked into the density and further refined interactively with MDFF.

Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome.
Thomas Becker, Jean-Paul Armache, Alexander Jarasch, Andreas M Anger, Elizabeth Villa, Heidemarie Sieber, Basma Abdel Motaal, Thorsten Mielke, Otto Berninghausen, and Roland Beckmann. Nat. Struct. Mol. Biol., 18, 715-720, 2011.

No-go decay (NGD) is a mRNA quality-control mechanism in eukaryotic cells that leads to degradation of mRNAs stalled during translational elongation. The key factors triggering NGD are Dom34 and Hbs1. We used cryo-EM to visualize NGD intermediates resulting from binding of the Dom34–Hbs1 complex to stalled ribosomes. At subnanometer resolution, all domains of Dom34 and Hbs1 were identified, allowing the docking of crystal structures and homology models. Moreover, the close structural similarity of Dom34 and Hbs1 to eukaryotic release factors (eRFs) enabled us to propose a model for the ribosome-bound eRF1–eRF3 complex. Collectively, our data provide structural insights into how stalled mRNA is recognized on the ribosome and how the eRF complex can simultaneously recognize stop codons and catalyze peptide release.

Correcting and preventing stereochemical errors

A recent publication at BMC Bioinformatics describes two VMD plugins that can be used to detect, visualize, and correct stereochemical errors in macromolecular structures. The plugins can also be used to generate restraints that prevent chirality and peptide bond configuration errors from arising in simulations where high forces are applied, such as MDFF simulations. Use of the plugins is described in the Structure Check Tutorial.

Stereochemical errors and their implications for molecular dynamics simulations.
Eduard Schreiner, Leonardo G. Trabuco, Peter L. Freddolino, and Klaus Schulten. BMC Bioinformatics, 12, 190, 2011.

Background: Biological molecules are often asymmetric with respect to stereochemistry, and correct stereochemistry is essential to their function. Molecular dynamics simulations of biomolecules have increasingly become an integral part of biophysical research. However, stereochemical errors in biomolecular structures can have a dramatic impact on the results of simulations.

Results: Here we illustrate the effects that chirality and peptide bond configuration flips may have on the secondary structure of proteins throughout a simulation. We also analyze the most common sources of stereochemical errors in biomolecular structures and present software tools to identify, correct, and prevent stereochemical errors in molecular dynamics simulations of biomolecules.

Conclusions: Use of the tools presented here should become a standard step in the preparation of biomolecular simulations.

Ribosome-SecYEG visualized in a membrane environment

A sub-nanometer cryo-EM reconstruction of the ribosome-SecYEG complex has been reported in the latest issue of Nature Structural & Molecular Biology. MDFF was employed to interpret the cryo-EM data at atomic level. The work is a collaboration between the Schulten (University of Illinois at Urbana-Champaign, USA) and Beckmann (University of Munich, Germany) groups.

Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.
Jens Frauenfeld, James Gumbart, Eli O. van der Sluis, Soledad Funes, Marco Gartmann, Birgitta Beatrix, Thorsten Mielke, Otto Berninghausen, Thomas Becker, Klaus Schulten, and Roland Beckmann. Nat. Struct. Mol. Biol., 18, 614-621, 2011.

The ubiquitous SecY–Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome–lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor–gated PCC in the membrane.

Insights into translational fidelity

In a recent work published by the EMBO Journal, images of the ribosome bound to either a cognate or a near-cognate tRNA were obtained by cryo-electron microscopy, and MDFF was employed to generate atomic models, shedding light into the mechanisms by which the ribosome discriminates between correct and incorrect tRNAs. The work is a collaboration between the Schulten (University of Illinois at Urbana-Champaign, USA) and the Frank (Columbia University, USA) groups.

Structural insights into cognate vs. near-cognate discrimination during decoding.
Xabier Agirrezabala, Eduard Scheiner, Leonardo G. Trabuco, Jianlin Lei, Rodrigo F. Ortiz-Meoz, Klaus Schulten, Rachel Green, and Joachim Frank. EMBO J., 30, 1497-1507, 2011.

The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with (UGA/stop) codons and incubated with ternary complex containing the near-cognate Trp-tRNA{Trp} in the presence of kirromycin. Going through more than 350,000 images and employing image classification procedures, we find 8% in which the ternary complex is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to EF-Tu and the ribosome, as well as the domain movements of the ribosome. The data bring direct structural insights into the induced fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues as to how the conformational signals conveyed to the GTPase differ in the two cases.

Molecular dynamics of EF-G during translocation

A study published in Proteins has employed both traditional molecular dynamics simulations and MDFF to study the dynamics of elongation factor G (EF-G). The work is a collaboration between the Schulten (University of Illinois at Urbana-Champaign, USA) and the Frank (Columbia University, USA) groups.

Molecular dynamics of EF-G during translocation.
Wen Li, Leonardo G. Trabuco, Klaus Schulten, and Joachim Frank. Proteins, 79, 1478-1486, 2011.

Elongation factor G (EF-G) plays a crucial role in two stages of mRNA-(tRNA)2 translocation. First, EF-G•GTP enters the pretranslocational ribosome in its intersubunit- rotated state, with tRNAs in their hybrid (P/E, A/P) positions. Second, a conformational change in EF-G’s domain IV induced by GTP hydrolysis disengages the mRNA-anticodon stem-loops of the tRNAs from the decoding center to advance relative to the small subunit when the ribosome undergoes a backward inter-subunit rotation. These events take place as EF-G undergoes a series of large conformational changes as visualized by cryo-EM and X-ray studies. The number and variety of these structures leave open many questions on how these different configurations form during the dynamic translocation process. To understand the molecular mechanism of translocation, we examined the molecular motions of EF-G in solution by means of molecular dynamics simulations. Our results show: (1) rotations of the super-domain formed by domains III-V with respect to the super-domain formed by I-II, and rotations of domain IV with respect to domain III; (2) flexible conformations of both 503- and 575-loops; (3) large conformational variability in the bound form provided by the interaction between domain V and the GTPase-associated center; (4) after GTP hydrolysis, the switch I region seems to be instrumental for effecting the conformational change at the end of domain IV implicated in the disengagement of the codon-anticodon helix from the decoding center.

Applications of the MDFF method reviewed

A special edition of the Journal of Structural Biology has been published with the theme "Combining computational modeling with sparse and low-resolution data." The special edition includes an article reviewing the first applications of the MDFF method, providing also an assessment of the accuracy of MDFF models.

Applications of the molecular dynamics flexible fitting method.
Leonardo G. Trabuco, Eduard Schreiner, James Gumbart, Jen Hsin, Elizabeth Villa, and Klaus Schulten. J. Struct. Biol., 173, 420-427, 2011.

In recent years, cryo-electron microscopy (cryo-EM) has established itself as a key method in structural biology, permitting the structural characterization of large biomolecular complexes in various functional states. The data obtained through single-particle cryo-EM has recently seen a leap in resolution thanks to landmark advances in experimental and computational techniques, resulting in sub-nanometer resolution structures being obtained routinely. The remaining gap between these data and revealing the mechanisms of molecular function can be closed through hybrid modeling tools that incorporate known atomic structures into the cryo-EM data. One such tool, molecular dynamics flexible fitting (MDFF), uses molecular dynamics simulations to combine structures from X-ray crystallography with cryo-EM density maps to derive atomic models of large biomolecular complexes. The structures furnished by MDFF can be used subsequently in computational investigations aimed at revealing the dynamics of the complexes under study. In the present work, recent applications of MDFF are presented, including the interpretation of cryo-EM data of the ribosome at different stages of translation and the structure of a membrane-curvature-inducing photosynthetic complex.