Thursday, December 2, 2010

Models of ribosomes in complex with tmRNA obtained by MDFF

Two new back-to-back articles in the EMBO Journal describe atomic models of ribosomes in complex with tmRNA, a molecule that rescues the ribosomes stalled at truncated messages. MDFF was employed in both studies to aid the interpretation of the cryo-EM reconstructions.

tmRNA–SmpB: a journey to the centre of the bacterial ribosome.
FĂ©lix Weis, Patrick Bron, Emmanuel Giudice, Jean-Paul Rolland, Daniel Thomas, Brice Felden, and Reynald Gillet. EMBO J., 29, 3810-3818, 2010.

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA–SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA–SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA–SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA–SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.

Visualizing the transfer-messenger RNA as the ribosome resumes translation.
Jie Fu, Yaser Hashem, Iwona Wower, Jianlin Lei, Hstau Y Liao, Christian Zwieb, Jacek Wower, and Joachim Frank. EMBO J., 29, 3819-3825, 2010.

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable ‘arc and fork’ structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.

Thursday, November 18, 2010

Formation of salt bridges mediates internal dimerization of myosin VI medial tail domain

A new article by the Schulten group (University of Illinois at Urbana-Champaign, USA) employs MDFF in an novel way, namely to retrieve atomistic information from a coarse-grained model of myosin.

Formation of salt bridges mediates internal dimerization of myosin VI medial tail domain.
HyeongJun Kim, Jen Hsin, Yanxin Liu, Paul R. Selvin, and Klaus Schulten. Structure, 18, 1443-1449, 2010.

The unconventional motor protein, myosin VI, is known to dimerize upon cargo-binding to its C-terminal end. It has been shown that one of its tail domains, called the medial tail domain, is a dimerization region. The domain contains an unusual pattern of alternating charged residues and a few hydrophobic residues. To reveal the unknown dimerization mechanism of the medial tail domain, we employed molecular dynamics and single-molecule experimental techniques. Both techniques suggest that the formation of electrostatic-based inter-helical salt bridges between oppositely-charged residues is a key dimerization factor. For the dimerization to occur, the two identical helices within the dimer don't bind in a symmetric fashion, but rather with an off-set of about one helical repeat. Calculations of the dimer-dissociation energy find the contribution of hydrophobic residues to the dimerization process to be minor; they also find that the asymmetric homodimer state is energetically favorable over a state of separate helices.

Wednesday, October 13, 2010

Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide

Another MDFF application has been recently published by the Roland Beckmann group (University of Munich, Germany). Their publication describes two cryo-EM structures of eukaryotic ribosomes stalled by regulatory peptides.

Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide. Shashi Bhushan, Helge Meyer, Agata L. Starosta, Thomas Becker, Thorsten Mielke, Otto Berninghausen, Michael Sattler, Daniel N. Wilson, and Roland Beckmann. Mol. Cell, 40, 138-146, 2010.

Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes.

Wednesday, September 15, 2010

The actin-myosin interface

MDFF has recently been applied by Michael Lorenz and Kenneth C. Holmes (Max Planck Institute for Medical Research, Heidelberg, Germany) to obtain an atomic model of an actin-myosin complex. Here's the abstract of their publication:

The actin-myosin interface. Michael Lorenz and Kenneth C. Holmes. Proc. Natl. Acad. Sci. USA, 107, 12529-12534, 2010.

In order to understand the mechanism of muscle contraction at the atomic level, it is necessary to understand how myosin binds to actin in a reversible way. We have used a novel molecular dynamics technique constrained by an EM map of the actin-myosin complex at 13-A resolution to obtain an atomic model of the strong-binding (rigor) actin-myosin interface. The constraining force resulting from the EM map during the molecular dynamics simulation was sufficient to convert the myosin head from the initial weak-binding state to the strong-binding (rigor) state. Our actin-myosin model suggests extensive contacts between actin and the myosin head (S1). S1 binds to two actin monomers. The contact surface between actin and S1 has increased dramatically compared with previous models. A number of loops in S1 and actin are involved in establishing the interface. Our model also suggests how the loop carrying the critical Arg 405 Glu mutation in S1 found in a familial cardiomyopathy might be functionally involved.

Saturday, January 23, 2010

New VMD plugins for structure validation and correction

Anyone working on molecular modeling soon realizes that atomic structures sometimes contain errors. Some of these errors can generate quite dramatic effects when the structures are simulated using molecular dynamics techniques. For example, an incorrect cis peptide bond in an alpha helix that disrupts the hydrogen bonding network can lead to the unfolding of the helix. Furthermore, MD force fields do not contain terms that prevent changes in chirality, so simulations where large forces are applied may introduce chirality errors.

To address these issues, we have recently added two new plugins to the development version of VMD: cispeptide and chirality (written by Leonardo Trabuco and Eduard Schreiner). They can be used to identify, visualize, and fix certain structure errors. The plugins can also be used to prevent such errors from occurring in certain kinds of MD simulations, as exemplified in the MDFF tutorial. We just posted a new tutorial that explains how to use these new plugins.

The cispeptide plugin identifies all cis peptide bonds in a protein structure and displays them graphically. The user can change any of the cis peptide bonds to the more common trans configuration if needed. To achieve that while still providing a physically sound structure, an actual molecular dynamics simulation is performed from within VMD by making use of an updated version of the AutoIMD plugin, which interfaces with our molecular dynamics simulation software NAMD.

The chirality plugin works in a very similar way, identifying all unusual configuration in chiral centers of proteins and nucleic acids. The user can also easily display the identified errors and correct them from within VMD.

Please give these new plugins a try and let us know what you think. Enjoy!

Tuesday, January 12, 2010

MDFF now available for Mac

New VMD development builds were posted today. These builds fix a known problem with Tcl linkage that previously prevented MDFF from working on the Mac.

If you are a Mac user, give this latest version a try and let us know if you find any problems.

Friday, January 8, 2010

MDFF tutorial updated

The MDFF tutorial has been updated and now includes a section on how to perform flexible fitting in explicit solvent.

Wednesday, January 6, 2010

Welcome to the MDFF blog!

Here you will find all the news about the Molecular Dynamics Flexible Fitting (MDFF) method, which combines high-resolution structures with low-resolution density maps. We will post updates on software development, training material, research applications, and more.