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Proc Natl Acad Sci U S A. Jun 29, 2004; 101(26): 9595–9600.
Published online Jun 21, 2004. doi:  10.1073/pnas.0308667101
PMCID: PMC470720
Biochemistry

Crystal structure of elongation factor P from Thermus thermophilus HB8

Abstract

Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å resolution. EF-P consists of three β-barrel domains (I, II, and III), whereas eIF-5A has only two domains (N and C domains). Domain I of EF-P is topologically the same as the N domain of eIF-5A. On the other hand, EF-P domains II and III share the same topology as that of the eIF-5A C domain, indicating that domains II and III arose by duplication. Intriguingly, the N-terminal half of domain II and the C-terminal half of domain III of EF-P have sequence homologies to the N- and C-terminal halves, respectively, of the eIF-5A C domain. The three domains of EF-P are arranged in an “L” shape, with 65- and 53-Å-long arms at an angle of 95°, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses domain topologies different from those of the known tRNA-mimicry translation factors. Domain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N domain.

Translation elongation factor P (EF-P) was found as a protein that stimulates the peptidyltransferase activity of the 70S ribosome in Escherichia coli (1). EF-P enhances dipeptide synthesis with N-formylmethionyl-tRNA and puromycin in vitro, suggesting its involvement in the formation of the first peptide bond of a protein (2). E. coli EF-P is encoded by the efp gene and consists of 188 amino acid residues (3). The efp genes are universally conserved in Bacteria (4). Gene interruption experiments in E. coli revealed that the efp gene is essential for cell viability and is required for protein synthesis (3). The amount of EF-P in E. coli cells is ≈1/10th of that of EF-G; 800–900 molecules of EF-P exist in a cell, an amount consistent with 1 EF-P per 10 ribosomes (5).

EF-P reportedly binds to both the 30S and 50S ribosomal subunits (6). Antibiotic sensitivity and footprinting studies have indicated that EF-P binds near the streptomycin-binding site of the 16S rRNA in the 30S subunit (6). EF-P also interacts with domains II and V of the 23S rRNA, i.e., near the peptidyltransferase center (PTC) (6, 7). Ribosome reconstitution experiments have shown that the L16 ribosomal protein or its N-terminal 47-residue fragment was required for EF-P-mediated peptide bond synthesis, whereas L11, L15, or L7/L12 were not (810).

Eukarya and Archaea seem to lack EF-P, although a similar function may be mediated by eukaryotic initiation factor 5A (eIF-5A) (4, 6). The eIF-5A protein is composed of ≈140 amino acid residues and is shorter than EF-P by ≈40 residues. Complete intracellular depletion of eIF-5A results in cell growth inhibition; however, protein synthesis seems to be only slightly reduced (11). eIF-5A is a unique cellular protein that contains the unusual amino acid hypusine [Nε-(4-aminobutyl-2-hydroxy)-l-lysine], which is formed by posttranslational modification of a specific lysine residue. The enzyme that modifies lysine to hypusine in eIF-5A is essential for yeast viability (12). On the other hand, hypusine is not found in bacteria (13). The structures of eIF-5A from three Archaea, Methanococcus jannaschii, Pyrobaculum aerophilum, and Pyrococcus horikoshii, have two domains, which are composed of several β-strands (1416).

Crystallizations of EF-P from E. coli and Aquifex aeolicus have been reported (7, 17). In this article, we report the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å resolution. The EF-P structures are β-rich and is divided into three β-barrel domains (domains I, II, and III). Domains II and III of EF-P share a very similar topology. The structures of domains I and II of EF-P are superposable on the structures of the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5A proteins. The overall structure of EF-P is strikingly similar to the L-shaped structure of tRNA.

Materials and Methods

Protein Preparation and Crystallization. The DNA fragment encoding EF-P, the protein TT0860 (DNA Data Base in Japan, accession no. AB103477), was isolated from the T. thermophilus HB8 genome and was cloned into the expression plasmid, pET11a (Novagen). E. coli BL21(DE3) was transformed with the vector, and T. thermophilus EF-P was overexpressed. The protein was purified by successive chromatography steps on Q Sepharose and HiLoad Superdex 75 columns (Amersham Biosciences). Hampton Research Crystal Screen (18) was used to determine the initial crystallization conditions for EF-P. The final crystallization conditions, 100 mM Hepes-Na buffer (pH 7.6) and 1.35 M lithium sulfate at 16°C yielded high-quality crystals suitable for x-ray diffraction data collection. They belong to the space group P212121, with unit cell dimensions a = 55.8, b = 78.4, and c = 138.9 Å. The crystallographic asymmetric unit contains two nearly identical EF-P monomers.

Data Collection and Structure Determination. The crystal structure of EF-P was solved by the multiple isomorphous replacement method. Three heavy-atom derivatives were prepared by soaking the EF-P crystals for 12 h in reservoir solutions containing 2 mM potassium tetrachloroaurate (III), 2 mM sodium ethylmercurithiosalicylate, and 2 mM mersalyl acid, respectively (Table 1). All the native and heavy-atom derivative data sets were collected from frozen crystals at 90 K by using synchrotron radiation at the SPring-8 beam lines (Hyogo, Japan). The data were processed with the program hkl2000 (19). Determination of the heavy atom positions and calculation of the multiple isomorphous replacement phases were carried out by using the program solve (20). The experimental phases were improved by using the resolve program (20) and further refined by using the arp/warp program (21) to 1.65 Å. The improved electron density map was of high quality, which allowed the arp/warp program to automatically build an almost complete model of one of the EF-P monomers (molecule A) in the asymmetric unit. Because the structure of the other EF-P molecule in the asymmetric unit (molecule B) is practically identical with that of molecule A, the molecule B model was readily produced by fitting the molecule A model to the electron density. The models were manually adjusted to the electron density by using the o program (22). Because no clear electron density was observed for the loop region (amino acid residues 139–145) of molecule B, these residues were excluded from the coordinates. The refinement was carried out with several rounds of conventional molecular dynamics protocols with the cns program (23), with all data in the resolution range of 40–1.65 Å. The refinement converged to an R factor of 21.3% (Rfree = 24.9%) at a 1.65-Å resolution (Table 1). The final model has 91.8% and 8.2% of the amino acid residues in the most favored and additional allowed regions, respectively, of the Ramachandran plot, as indicated by the program procheck (24). Graphic figures were created with the programs molscript (25) and raster3d (26) or grasp (27).

Table 1.
Crystallographic data

Determination of Molecular Weight in Solution. The molecular weight of T. thermophilus EF-P in solution was estimated by light scattering (DynaPro 99, Protein Solutions, Charlottesville, VA) and by analytical ultracentrifugation (Optima XL-1, Beckman Coulter). Light scattering was performed at 20°C in 20 mM Tris·HCl buffer (pH 7.5) containing 150 mM NaCl and 1 mM DTT. Analytical ultracentrifugation was performed at 20°Cin20 mM Tris·HCl buffer (pH 7.5) containing 150 mM NaCl and 5 mM 2-mercaptoethanol.

Results and Discussion

Overall Structure. In the present study, we determined the crystal structure of EF-P from T. thermophilus at a 1.65-Å resolution by the multiple isomorphous replacement method. The crystallographic data are summarized in Table 1. The atomic coordinates have been deposited in the Protein Data Bank (PDB ID code 1UEB). In the crystal, two molecules (A and B) are in the asymmetric unit (Fig. 1A). The EF-P protein is a β-rich protein containing 16 β-strands and is made up of three β-barrel domains (domains I, II, and III) (Fig. 1B).

Fig. 1.
The structure of T. thermophilus EF-P. (A) Ribbon diagram showing the two molecules (A and B) in the asymmetric unit. The arrows represent β-strands. The three domains of each molecule are colored green, red, and blue, in dark and light tones ...

The β-strands of molecule A are designated as β1–β16, whereas the corresponding β-strands of molecule B are β1′–β16′, to discriminate between the two molecules. In the protein crystal, the β3-strand forms an antiparallel β-sheet with the β3′-strand of the other monomer (Fig. 1 A). This interaction connects the two monomers in the asymmetric unit, to form the dimer. A large, flat, six-stranded β-sheet is formed by β3, β4, β5, β3′, β4′, and β5′. The two monomers are related by a pseudo 2-fold axis, which is perpendicular to the β-sheet and passes near the carbonyl oxygen of His-27 on β3 (or β3′). The buried surface area between the EF-Ps was 539 Å2, which is ≈5% of the monomer surface of EF-P. The T. thermophilus EF-P exists as a monomer (20,224 Da) under physiological conditions, as shown by analytical ultracentrifugation (20.8 kDa) and light-scattering experiments (23.2 kDa) (Fig. 2). This finding suggests that the monomer is the major functional unit of EF-P, although we cannot exclude the possibility that EF-P dimerizes during some functional stage.

Fig. 2.
A plot of the sedimentation equilibrium data with the residuals from the best fit to a single ideal species. This plot shows the data with protein at 0.5 mg·ml-1 and a speed of 20,000 rpm. The estimated partial specific volume of the protein is ...

The overall shape of the EF-P monomer (Figs. (Figs.1C1C and 3 A and B) is remarkably similar to the L shape of the tRNA molecule (Fig. 3C). One arm of the L, made by domains I and II (Fig. 1C), is ≈65 Å long and 23 Å wide (Fig. 3 A and B), whereas the other arm, formed by domains II and III (Fig. 1C), is ≈53 Å long and 25 Å wide (Fig. 3 A and B). The angle made by the two arms of EF-P is ≈95° (Fig. 3 A and B). In the yeast tRNAPhe L-shaped structure, with two arms at ≈90° (2830), the acceptor-T arm is ≈65 Å long and 22 Å wide, whereas the anticodon-D arm is ≈70 Å long and 20 Å wide (Fig. 3C). The overall shapes and the sizes of tRNA molecules are well conserved. The shape and the size of the EF-P molecule are similar to those of tRNA molecules. Notably, EF-P is an acidic protein (calculated pI = 4.6), and most of its surface is negatively charged (Fig. 3 A and B). Therefore, its overall shape is reminiscent of that of tRNA, although it is currently unclear which arm of EF-P corresponds to the acceptor or anticodon arm of tRNA (Fig. 3 A and B).

Fig. 3.
Structure comparison of EF-P with tRNA and ribosome-binding proteins. (A and B) EF-P from T. thermophilus (PDB ID code 1UEB). (C) tRNAPhe from Saccharomyces cerevisiae (PDB ID code 1EVV). (D) EF-G from T. thermophilus (PDB ID code 1EFG). (E) Ribosome ...

The structures of the two EF-P monomers in the asymmetric unit are quite similar to each other, with an rms deviation (rmsd) of 0.79 Å over all the protein atoms. Nevertheless, their interdomain orientations differ slightly. Although the domain I structures of molecules A and B are practically the same (they can be superposed on each other with an rmsd of 0.57 Å), the relative orientation of domain I to domain II in the two molecules differs by ≈4°. Therefore, this arm is slightly flexible. On the other hand, the difference in the relative orientation of domains II and III between molecules A and B is negligibly small. Both of the domain I–II and II–III interfaces are formed by hydrophobic side chains with high surface complementarities. Therefore, the L shape of EF-P is likely to be a native conformation, rather than an artifact due to crystal packing.

Several proteins possess domain(s) similar to a portion of tRNA. The C-terminal domain of EF-G, protruding from the globular GTPase domain, reportedly has a shape similar to that of the anticodon-stem loop in the EF-Tu–tRNA–GDPNP ternary complex (31, 32). Ribosome recycling factor, eukaryal release factor 1, and release factor 2 each possess a protruding domain (3337). The entire structure of EF-P mimics the overall shape of a tRNA molecule, like these tRNA-mimicking proteins (Fig. 3).

However, the “tRNA mimicry” does not necessarily mean that the proteins bind to the tRNA-binding sites on the ribosome as tRNA molecules do (36). Based on a cryo-electron microscopy analysis, it is reported that release factor 2 is incorporated in the ribosome by assuming a different shape from that observed in the crystal structure and by changing its interdomain orientations (38, 39). A biochemical analysis revealed that the binding mode of ribosome-recycling factor is different from that of tRNA (40). It has been hypothesized that the tRNA mimicry might allow the proteins to pass through the entrance of the ribosome (36). In this context, biochemical data suggested that EF-P binds to the A site of the ribosome (6, 7). Ganoza et al. (6) proposed that the EF-P-binding domain is very near the site of EF-Tu and EF-G binding on both the 30S and 50S subunits. However, the ribosome-binding manner of EF-P is unknown and must be studied further with the ribosome-bound state structure of the EF-P.

Domain Architectures. The N-terminal domain I (amino acid residues 1–64) contains six β-strands (β1–β6) and a one-turn 310-helix (Fig. 1 B and C). The strands β2, β3, β4, and β5 form a large antiparallel β-sheet, whereas β1 and β6 form a smaller, curved antiparallel β-sheet. The larger β-sheet is flat on one side, but its other side is curved, and, together with the smaller β-sheet, it is involved in a β-barrel with a hydrophobic core. The 310-helix is located between β1 and β2, forming the lid of the barrel. The β3-, β4-, and β5-strands are much longer than the other strands, and therefore, the region connecting β3 and β4 (amino acid residues 28–36) protrudes outward. The conserved basic residues, Arg-8, Lys-29, Arg-32, Lys-40, and Lys-42, are clustered on the β3-side surface of domain I (Fig. 3A), implying the significance of this region in EF-P function, such as nucleic acid binding. On the other hand, the opposite surface of domain I (Fig. 3B) is more negatively charged, as Asp-6, Asp-16, and Glu-61 are highly conserved among the EF-P sequences (Fig. 4A).

Fig. 4.
Alignment of the amino acid sequences of EF-P and eIF-5A. bTth, bEco, and bBsu are the bacterial EF-P proteins from T. thermophilus, E. coli, and Bacillus subtilis, respectively. eHum, eSce, ePae, and eMja are the eIF-5A proteins from Homo sapiens, S. ...

It is remarkable that central domain II and C-terminal domain III of EF-P possess the same fold (Fig. 1 B and C). The two β-barrel domains are tandemly arranged along the axis of one arm of the L-shaped EF-P molecule. Domain II (amino acid residues 65–126) contains five β-strands (β7–β11). The β7-, β8-, and β9-strands form a curved antiparallel β-sheet on one hand, whereas β7, β10, and β11 form a similar antiparallel β-sheet on the other, and thus together they construct a typical β-barrel structure with a hydrophobic core. Domain III (amino acid residues 127–184) also possesses a β-barrel architecture consisting of five β-strands (β12–β16), in which two antiparallel β-sheets, consisting of β12, β13, and β14, and β12, β15, and β16, respectively, face each other. Thus, the two domains possess the same topology for the strand connectivities. Domains II and III were superposed on each other with an rmsd of 1.2 Å for 31 Cα atoms. These domains share partial sequence similarity (10 of 58 residues are identical), which implies that they originated from a single domain, probably by a duplication event. It should be noted here that the sequence of domain III is much better conserved than that of domain II in the EF-P proteins (Fig. 4).

According to a DALI-based protein–structure comparison, the fold composed of domains II and III is similar to that of the so-called oligonucleotide-binding fold, observed in E. coli coldshock protein, the RNA-binding domains of E. coli polyribonucleotide nucleotidyltransferase, E. coli transcription factor Rho, Pyrococcus kodakaraensis aspartyl-tRNA synthetases, and so forth. Thus, it is possible that domains II and/or III of EF-P are involved in RNA (or DNA) binding. However, because almost the entire surface of domain II is negatively charged (Glu-76, Glu-78, Asp-84, Glu-89, and Glu-106 are conserved), this domain probably does not bind nucleic acids. In contrast, one surface of domain III has a patch of conserved basic residues, including Arg-140, Lys-149, Arg-176, and Arg-183, which are probably favorable for nucleic acid binding. The other surface of domain III is negatively charged, and Asp-134, Glu-154, Glu-166, and Glu-169 are conserved.

Comparison Between EF-P and eIF-5A. eIF-5A is an archaeal/eukaryal paralog of EF-P. Thus far, three eIF-5A crystal structures have been reported, from M. jannaschii, P. aerophilum, and P. horikoshii (1416). These structures revealed that eIF-5A consists of only two β-barrel domains. These N and C domains appear to correspond to domains I and II (or III), respectively, of EF-P. The overall shape of the two-domain eIF-5A is a straight bar, in contrast to the L-shaped structure of the threedomain EF-P (Fig. 5A). Intriguingly, slight flexibility in the relative orientation of domain I to domain II has also been found for the M. jannaschii and P. horikoshii eIF-5A proteins (≈7°) (14, 16), which is very similar to the internal-domain flexibility of EF-P (≈4°) described above.

Fig. 5.
Structure comparison of EF-P and eIF-5A. (A) Superimposition of the ribbon diagrams of T. thermophilus EF-P (blue) and M. jannaschii eIF-5A (yellow). (B) Amino acid residues conserved in EF-Ps and eIF-5As color-coded on the surface of T. thermophilus ...

The structure of the EF-P domain I superposed well on those of the N domains of M. jannaschii eIF-5A (rmsd = 1.3 Å per 61 Cα atoms), P. aerophilum eIF-5A (rmsd = 1.2 Å per 60 Cα atoms), and P. horikoshii eIF-5A (rmsd = 1.4 Å per 63 Cα atoms). Consistent with the structural information, the domain I amino acid sequences of the known EF-P and eIF-5A proteins share some similarity (Fig. 4). According to a structure-based sequence comparison, 42% of the T. thermophilus EF-P amino acid residues are conserved or semiconserved in eIF-5As. In particular, the amino acid residues corresponding to Lys-29, Gly-31, Gly-33, and Ala-35 of the T. thermophilus EF-P are absolutely conserved in the EF-P/eIF-5A superfamily, and they are located on the loop connecting β3 and β4 in both the EF-P and eIF-5A structures. The eIF-5As conserve a Lys residue at the tip of the loop (Lys-40, Lys-42, and Lys-37 for the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5A proteins, respectively). This Lys residue is modified posttranslationally to a hypusine to generate the mature eIF-5A (12). It is remarkable that a Lys or Arg residue is also strictly conserved at the corresponding position of the EF-P proteins in bacteria (Fig. 4). For T. thermophilus EF-P, the corresponding basic residue is Arg-32. In the present EF-P crystal structure, the Arg-32 side chain protrudes toward the solvent at the end of the domain I arm. The bacterial EF-P reportedly lacks hypusine (13). Nevertheless, the conservation of the amino acid residues in the β3–β4 connective linker and the basic residue at the tip of the loop implies the significance of this region for EF-P function.

The eIF-5A C domain was compared with the EF-P domain II (Fig. 5A). The C domains of the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5As overlapped well on the EF-P domain II, with rmsd values of 1.4 Å, 1.5 Å, and 2.0 Å, for 53, 58, and 56 Cα atoms, respectively. The similarity between the EF-P and eIF-5A sequences exists only in the N-terminal half of domain II (the β7–β9 region in EF-P) and not in the C-terminal half of domain II (Fig. 3). On the other hand, as the EF-P domains II and III share the same folding topology, the eIF-5A domain II also superposed well on the EF-P domain III (rmsd values of 2.1 Å, 2.4 Å, and 2.2 Å, for 41, 41, and 43 Cα atoms of the eIF-5A proteins from M. jannaschii, P. aerophilum, and P. horikoshii, respectively). It is remarkable that the amino acid sequence of the C-terminal half of the eIF-5A C domain is similar to that of the C-terminal half of EF-P domain III (the β15–β16 region in EF-P) (Fig. 5B). Thus, a gap appears in the eIF-5A sequences, as compared with the EF-P sequences. This implies the possibility that eIF-5A originated from an ancestral three-domain protein common to EF-P by a deletion event, in which the ancestral eIF-5A might have lost the region corresponding to the EF-P β10–β14 region. Because the missing region topologically corresponds to a single β-barrel domain, the resultant eIF-5A C domain retains the same folding topology as domains II and III of EF-P. On the other hand, it is also possible that the sequence of the EF-P β10–β14 region diversified after domains II and III were formed by duplication.

Concluding Remarks. The overall tRNA-like shape of the EF-P molecule and its charge distribution seem to be suitable for this protein to bind to the ribosome by spanning the two subunits (6). EF-P may bind to the tRNA-binding site(s) on the ribosome by mimicking the tRNA shape. eIF-5A corresponds to domains I and II of EF-P. It is interesting to note that, in this context, eIF-5A might correspond to a minihelix or anticodon helix of tRNA. Although eIF5A is not thought to be essentially involved in translation in yeast (11), it is also possible that, in Archaea and Eukarya, some other protein or RNA factor(s) compensates structurally or functionally for the missing third domain. Many questions about EF-P still remain. How does EF-P interact with the ribosome? Which arm of the L corresponds to the acceptor or anticodon arm of tRNA? Does it form a ternary complex with EF-Tu·GTP? How can it activate the peptidyltransferase of the ribosome? To answer these questions, further functional and structural studies are needed.

Acknowledgments

We thank R. Ushikoshi, H. Tanaka, and Y. Kamewari for preparation of the T. thermophilus EF-P protein, H. Nakajima and Drs. Y. Kawano and N. Kamiya for supporting our data collection at beamline 45PX at SPring-8, and Dr. S. Yokobori (Tokyo University of Pharmacy and Life Science) for helpful discussions about the phylogenetic analysis. This work was supported in part by a grant from the Organized Research Combination System of the Science and Technology Agency of Japan and by the RIKEN Structural Genomics/Proteomics Initiative and the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan.

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: EF, elongation factor; eIF-5A, eukaryotic initiation factor 5A; rmsd, rms deviation.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1UEB).

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