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Biomedical subjects

Joachim Frank

Publications and source records attributed to Joachim Frank.

15 recordsLinked to original sources
PubMed2003

Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy.

Combining structural data for the ribosome from x-ray crystallography and cryo-electron microscopy with dynamic models based on elastic network normal mode analysis, an atomically detailed picture of functionally important structural rearrangements that occur during translocation is elucidated. The dynamic model provides a near-atomic description of the ratchet-like rearrangement of the 70S ribosome seen in cryo-electron microscopy, and permits the identification of bridging interactions that either facilitate the conformational switching or maintain structural integrity of the 50S/30S interface. Motions of the tRNAs residing in the A and P sites also suggest the early stages of tRNA translocation as a result of this ratchet-like movement. Displacement of the L1 stalk, alternately closing and opening the intersubunit space near the E site, is observed in the dynamic model, in line with growing experimental evidence for the role of this structural component in facilitating the exiting of tRNA. Finally, a hinge-like transition in the 30S ribosomal subunit, similar to that observed in crystal structures of this complex, is also manifest as a dynamic mode of the ribosome. The coincidence of these dynamic transitions with the individual normal modes of the ribosome and the good correspondence between these motions and those observed in experiment suggest an underlying principle of nature to exploit the shape of molecular assemblies such as the ribosome to provide robustness to functionally important motions.

Binding Sites↗
PubMed2003

Locking and unlocking of ribosomal motions.

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.

Binding Sites↗
PubMed2003

Study of the structural dynamics of the E coli 70S ribosome using real-space refinement.

Cryo-EM density maps showing the 70S ribosome of E. coli in two different functional states related by a ratchet-like motion were analyzed using real-space refinement. Comparison of the two resulting atomic models shows that the ribosome changes from a compact structure to a looser one, coupled with the rearrangement of many of the proteins. Furthermore, in contrast to the unchanged inter-subunit bridges formed wholly by RNA, the bridges involving proteins undergo large conformational changes following the ratchet-like motion, suggesting an important role of ribosomal proteins in facilitating the dynamics of translation.

Bacterial Proteins↗
PubMed2003

Visualizing tmRNA entry into a stalled ribosome.

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.

Alanine↗
PubMed2003

A cryo-electron microscopic study of ribosome-bound termination factor RF2.

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.

Binding Sites↗
PubMed2003

Electron microscopy of functional ribosome complexes.

Cryoelectron microscopy has made a number of significant contributions to our understanding of the translation process. The method of single-particle reconstruction is particularly well suited for the study of the dynamics of ribosome-ligand interactions. This review follows the events of the functional cycle and discusses the findings in the context provided by the recently published x-ray structures.

Binding Sites↗
PubMed2003

An approach to examining model dependence in EM reconstructions using cross-validation.

Reference bias refers to a common problem in fitting experimental data to an initial model. Given enough free parameters, a good fit of any experimental data to the model can be obtained, even if the experimental data contain only noise. Reference-based alignment methods used in electron microscopy (EM) are subject to this type of bias, in that images containing pure noise can regenerate the reference. Cross-validation is based on the idea that the experimental data used to assess the validity of the fitting should not be the same data as were used to do the fitting. Here we present the application of cross-validation to one form of reference-based alignment: 3D-projection matching in single-particle reconstructions. Our results show that reference bias is indeed present in reconstructions, but that the effect is small for real data compared to that for random noise, and that this difference in behavior is magnified, rather than diminished, during iterative refinement.

Escherichia coli↗
PubMed2003

A twisted tRNA intermediate sets the threshold for decoding.

Putting together consistent cryo-EM structure, transient kinetic and mutant tRNA suppressor data, it appears that a deformed or waggling aminoacyl-tRNA is the critical transitional structure examined by the ribosome during decoding at the A site. The unusual conformation may be required for effective proofreading of the codon-anticodon complex.

Escherichia coli↗
PubMed2002

Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Anticodon↗
PubMed2002

An assay for local quality in cryo-electron micrographs of single particles.

High quality of the cryo-electron micrographs is of crucial importance for the success of single particle three-dimensional reconstruction methods. In analyzing some micrographs from cryo-electron microscopy specimens, we found an extraordinary variability, within the same micrograph, in the appearance of particles. We developed a method for analyzing the variability of local image quality, using correspondence analysis of local power spectra. With this technique, we discovered a strong systematic variation of the envelope modulating an otherwise unchanged contrast transfer function. The underlying causes may be uncontrollable effects, such as variations in the thickness of ice, instability of the holey carbon, and charging. The method of assaying, resulting in "local quality maps", may be useful as a general tool for screening micrographs used as input for reconstructions.

Cryoelectron Microscopy↗
PubMed2001

Single-particle imaging of macromolecules by cryo-electron microscopy.

Cryo-electron microscopy (cryo-EM) of biological molecules in single-particle (i.e., unordered, nonaggregated) form is a new approach to the study of molecular assemblies, which are often too large and flexible to be amenable to X-ray crystallography. New insights into biological function on the molecular level are expected from cryo-EM applied to the study of such complexes "trapped" at different stages of their conformational changes and dynamical interactions. Important molecular machines involved in the fundamental processes of transcription, mRNA splicing, and translation are examples for successful applications of the new technique, combined with structural knowledge gained by conventional techniques of structure determination, such as X-ray crystallography and NMR.

Animals↗
PubMedDate not supplied

Three-dimensional imaging of biological complexity.

Over the past 5 years, thanks to advances in both instrumentation and computational speed, three-dimensional imaging techniques using the electron microscope have been greatly improved in two areas: electron tomography of cell organelles or cell sections and reconstruction of macromolecules from single particles. Ice embedment has brought a breakthrough in the degree of preservation of specimens under close-to-native conditions. The current challenge is to push the resolution of electron tomographic imaging to a point where macromolecular signatures can be recognized within the cellular context. We show first progress toward this goal by examples in two areas of application: the structure of the muscle triad junction and the architecture and fine structure of mitochondria. As techniques of cryo-microtomy are perfected, we hope to be able to apply tomography to high-pressure frozen sections of tissue.

Animals↗
PubMedDate not supplied

Electron tomographic analysis of frozen-hydrated tissue sections.

Electron tomography of frozen-hydrated tissue sections enables analysis of the 3-D structure of cell organelles in situ and in a near-native state. In this study, 160-200-nm-thick sections were cut from high-pressure frozen rat liver, and improved methods were used for handling and mounting the sections. Automated data collection facilitated tilt-series recording at low electron dose (approximately 4000 e(-)/nm(2) at 400 keV). Higher doses (up to 10,000 e(-)/nm(2)) were found to increase contrast and smooth out surface defects, but caused section distortion and movement, with likely loss of high-resolution information. Tomographic reconstruction showed that knife marks were 10-40 nm deep and located on the "knife face" of the section, while crevices were 20-50 nm deep and found on the "block face." The interior of the section was normally free of defects, except for compression, and contained useful structural information. For example, the topology of mitochondrial membranes in tissue was found to be very similar to that in frozen-hydrated whole mounts of isolated mitochondria. In rare cases, a 15-nm banding pattern perpendicular to the cutting direction was observed in the interior of the section, most evident in the uniformly dense, protein-rich material of the mitochondrial matrix.

Animals↗