The MDFF Blog

Structural prediction combined with MDFF

2011-10-11T01:26:00.000-07:00

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

2011-08-08T06:45:00.000-07:00

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

2011-08-01T06:23:00.000-07:00

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

2011-05-04T09:01:00.000-07:00

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

2011-05-04T08:39:00.000-07:00

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

2011-04-27T03:29:00.000-07:00

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

2011-02-14T01:33:00.000-08:00

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.

Atomic models of an eukaryotic ribosome

2011-01-10T00:52:00.000-08:00

Two new back-to-back articles in PNAS describe atomic models of eukaryotic ribosomes, based on a cryo-EM reconstruction of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome. MDFF was employed to refine the atomic models, in particular an interactive version that leverages the IMD interface in VMD.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution.
Jean-Paul Armache, Alexander Jarasch, Andreas M. Anger, Elizabeth Villa, Thomas Becker, Shashi Bhushan, Fabrice Jossinet, Michael Habeck, Gülcin Dindar, Sibylle Franckenberg, Viter Marquez, Thorsten Mielke, Michael Thomm, Otto Berninghausen, Birgitta Beatrix, Johannes Söding, Eric Westhof, Daniel N. Wilson, and Roland Beckmann Proc. Natl. Acad. Sci. USA, 107, 19748-19753, 2011.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution. This map, together with a 6.1-Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES–ES and r-protein–ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Localization of eukaryote-specific ribosomal proteins in a 5.5Å cryo-EM map of the 80S eukaryotic ribosome.
Jean-Paul Armache, Alexander Jarasch, Andreas M. Anger, Elizabeth Villa, Thomas Becker, Shashi Bhushan, Fabrice Jossinet, Michael Habeck, Gülcin Dindar, Sibylle Franckenberg, Viter Marquez, Thorsten Mielke, Michael Thomm, Otto Berninghausen, Birgitta Beatrix, Johannes Södina, Eric Westhof, Daniel N. Wilson, and Roland Beckmann. Proc. Natl. Acad. Sci. USA, 107, 19754-19759, 2011.

Protein synthesis in all living organisms occurs on ribonucleoprotein particles, called ribosomes. Despite the universality of this process, eukaryotic ribosomes are significantly larger in size than their bacterial counterparts due in part to the presence of 80 r proteins rather than 54 in bacteria. Using cryoelectron microscopy reconstructions of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Å resolution, together with a 6.1-Å map of a translating Saccharomyces cerevisiae 80S ribosome, we have localized and modeled 74/80 (92.5%) of the ribosomal proteins, encompassing 12 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function, and evolution of the eukaryotic translational apparatus.

Models of ribosomes in complex with tmRNA obtained by MDFF

2010-12-02T01:07:00.000-08:00

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.
Félix 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.

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

2010-11-18T01:29:00.000-08:00

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.

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

2010-10-13T01:14:00.000-07:00

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.

The actin-myosin interface

2010-09-15T00:10:00.000-07:00

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.

New VMD plugins for structure validation and correction

2010-01-23T15:49:00.000-08:00

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!

MDFF now available for Mac

2010-01-12T15:00:00.000-08:00

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.

MDFF tutorial updated

2010-01-08T10:33:00.000-08:00

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

Welcome to the MDFF blog!

2010-01-06T12:25:00.000-08:00

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.