Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 Å resolution by electron cryomicroscopy and angular reconstitution † 1 †This article is dedicated to the memory of Anneke van Heel. 1Edited by M.F. Moody

Orlova, Elena V.; Dube, Prakash; Harris, J. Robin; Beckman, E.; Zemlin, F.; Markl, Jürgen; Heel, Marin van

doi:10.1006/jmbi.1997.1182

Cited by 200 publications

(162 citation statements)

References 78 publications

(88 reference statements)

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“…The resolution of this map is Ϸ3 nm according to the Fourier shell correlation criterion (Fig. 4), where the 3 threshold is corrected for C4 symmetry (28).…”

Section: Resultsmentioning

confidence: 99%

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Fonseca¹,

Morris²,

Nerou³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Materials and Methods Purification of IP3R.A membrane fraction was prepared from rat cerebella (17), resuspended (50 mg͞ml in 50 mM Tris͞1 mM EDTA, pH 8.3), and solubilized by the addition of 1% Triton X-100. After centrifugation (100,000 ϫ g for 60 min), the supernatant, supplemented with 250 mM NaCl, was loaded onto an Econopac heparin column (1 ml, Bio-Rad), washed with 25 ml of medium A (50 mM Tris͞250 mM NaCl͞10% glycerol, pH 8.3), supplemented with 1 mM EDTA, 1% Surfact-Amps X-100 (Pierce), and then with medium A supplemented with 0.1% Surfact-Amps X-100 (50 ml). IP 3 R was eluted from the heparin column directly onto a 2-ml Con A-agarose column (Sigma) by using medium A supplemented with 0.1% Surfact-Amps X-100 and 50 M decavanadate (18). After washing with 150 ml of medium B (50 mM Tris͞100 mM NaCl͞10% glycerol͞0.1% Surfact-Amps X-100, pH 8.3), IP 3 R was eluted by overnight incubation with 4 ml of medium B containing 800 mM methyl mannopyranoside (17). To assess the quaternary structure of the purified receptor, 0.2-ml fractions of the final eluate were loaded onto linear 10-30% sucrose gradients (10 ml) in 50 mM Tris͞1 mM EDTA͞0.1% Surfact-Amps X-100, pH 8.3, and centrifuged (134,000 ϫ g for 60 min). Gradients were calibrated by using a high-molecular-weight gel filtration standards kit (Amersham Biosciences). Size-exclusion HPLC was preformed by using a Zorbax GF-450 column (Jones Chromatography, Lakewood, CO) coupled to a Kontron system (Kontron Instruments, Watford, U.K.). All media included the following mixture of protease inhibitors: 150 nM aprotinin, 1 M pepstatin, 20 g͞ml soybean trypsin inhibitor, 1 mM benzamidine, 250 M PMSF, 250 M captopril, and 1 M bestatin.

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“…The resolution of this map is Ϸ3 nm according to the Fourier shell correlation criterion (Fig. 4), where the 3 threshold is corrected for C4 symmetry (28).…”

Section: Resultsmentioning

confidence: 99%

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Fonseca¹,

Morris²,

Nerou³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…The data converged after several rounds of iterative refinement in this manner, whereby roughly 40% of the class averages were discarded after assessment through cross-correlation functions. The resolution of the final three-dimensional map was determined by Fourier shell correlation (FCS) between two independent three-dimensional reconstructions (26) compensated for the C3 symmetry used (27).…”

Section: Methodsmentioning

confidence: 99%

Three-dimensional Model and Characterization of the Iron Stress-induced CP43′-Photosystem I Supercomplex Isolated from the Cyanobacterium Synechocystis PCC 6803

Bibby¹,

Nield

Barber

2001

Journal of Biological Chemistry

107

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The cyanobacterium Synechocystis PCC 6803 has been subjected to growth under iron-deficient conditions. As a consequence, the isiA gene is expressed, and its product, the chlorophyll a-binding protein CP43, accumulates in the cell. Recently, we have shown for the first time that 18 copies of this photosystem II (PSII)-like chlorophyll a-binding protein forms a ring around the trimeric photosystem I (PSI) reaction center (Bibby, T. S., Nield, J., and Barber, J. (2001) Nature, 412, 743-745). Here we further characterize the biochemical and structural properties of this novel CP43-PSI supercomplex confirming that it is a functional unit of approximately 1900 kDa where the antenna size of PSI is increased by 70% or more. Using electron microscopy and single particle analysis, we have constructed a preliminary three-dimensional model of the CP43-PSI supercomplex and used it as a framework to incorporate higher resolution structures of PSI and CP43 recently derived from x-ray crystallography. Not only does this work emphasize the flexibility of cyanobacterial lightharvesting systems in response to the lowering of phycobilisome and PSI levels under iron-deficient conditions, but it also has implications for understanding the organization of the related chlorophyll a/b-binding Pcb proteins of oxychlorobacteria, formerly known as prochlorophytes.Iron is the most abundant transition metal in the crust of the earth and is an absolute requirement for photosynthetic organisms such as cyanobacteria, because it is needed for many of the redox reactions of the photosynthetic electron transport system. However, in most aquatic ecosystems it can be sufficiently low to limit photosynthetic activity (1, 2). This finding is attributed mainly to the low solubility of Fe 3ϩ above neutral pH in oxygenic ecosystems (3). As a result, cyanobacteria and other microorganisms have evolved a number of responses to cope with frequently occurring conditions of iron deficiency (4). One such response is to express two "iron stress-induced" genes, isiA and isiB (5, 6), which are located on the same operon. The isiB gene encodes for flavodoxin, which can functionally replace the iron containing ferredoxin (7). The isiA gene encodes for a protein often called CP43Ј, because it has an amino acid sequence homologous to that of the chlorophyll a-binding protein, CP43 of photosystem II (PSII) 1 (8, 9). Like CP43, CP43Ј is predicted to have six transmembrane helices, and judged by the conservation of histidine residues, it is likely to bind the same number of chlorophyll a molecules. The major difference is that CP43Ј lacks the large hydrophilic loop that joins the luminal ends of helices V and VI of CP43. For this reason, it has 342 amino acids rather than 472 (see Fig. 1).Under iron stress, the isiA gene is transcribed into two messages, a monocistronic message containing only isiA and a dicistronic message that also contains the isiB gene (10). Although the discovery of the CP43-like iron stress-induced protein was made some time ago (11), its precise...

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“…18), was 12.4 Å (or 9.1 Å by the 3 criterion; see ref. 15) for the control, and 12.0 Å (or 8.4 Å by 3 ) for the ribosome-RRF complex. The falloff of the Fourier amplitudes toward higher spatial frequencies was corrected as described (18), using the small-angle x-ray solution scattering intensity distribution of the E. coli 70S ribosome.…”

mentioning

confidence: 99%

“…Here we present a three-dimensional cryo-electron microscopy (cryo-EM) map of the E. coli 70S ribosome-RRF complex at 12-Å resolution (8.4 Å by 3 criterion, see ref. 15). Our map directly shows a density attributable to RRF, in general agreement with the overall orientation of RRF inferred from the HRP study (14).…”

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confidence: 99%

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

Agrawal

Sharma

Kiel

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

148

170

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After the termination step of protein synthesis, a deacylated tRNA and mRNA remain associated with the ribosome. The ribosomerecycling factor (RRF), together with elongation factor G (EF-G), disassembles this posttermination complex into mRNA, tRNA, and the ribosome. We have obtained a three-dimensional cryo-electron microscopic map of a complex of the Escherichia coli 70S ribosome and RRF. We find that RRF interacts mainly with the segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. The binding of RRF induces considerable conformational changes in some of the functional domains of the ribosome. As compared to its binding position derived previously by hydroxyl radical probing study, we find that RRF binds further inside the intersubunit space of the ribosome such that the tip of its domain I is shifted (by Ϸ13 Å) toward protein L5 within the central protuberance of the 50S subunit, and domain II is oriented more toward the small ribosomal subunit (30S). Overlapping binding sites of RRF, EF-G, and the P-site tRNA suggest that the binding of EF-G would trigger the removal of deacylated tRNA from the P site by moving RRF toward the ribosomal E site, and subsequent removal of mRNA may be induced by a shift in the position of 16S rRNA helix 44, which harbors part of the mRNA. R ibosomes are responsible for translating genetic information carried by mRNAs into specific sequences of amino acids. Translation on the ribosome comprises of four main steps: (i) initiation, (ii) elongation, (iii) termination, and (iv) recycling. Recent advancements in structural studies of the translational machinery have helped elucidate binding positions and functions of various translational factors involved in various stages of initiation (1, 2), elongation (3, 4), and termination (5-7). The fourth step of translation requires binding of a dedicated protein factor, the ribosome-recycling factor (RRF), which in conjunction with elongation factor G (EF-G) helps removing the mRNA and last deacylated tRNA from the ribosome (see ref. 8).Atomic structures of RRF determined from five different species, including Escherichia coli, show that it is comprised of two structural domains: domain I, consisting of three long ␣-helix bundles, and the smaller domain II, which is an ␣͞␤ motif (9-13). Different orientations of domain II in these structures have been attributed to interdomain flexibility, which is thought to be necessary for RRF to function on the ribosome (12).The overall match in dimensions between RRF and tRNA (9) prompted the proposal of structural and functional molecular mimicry between the two molecules (9). In a recent study (14) using the hydroxyl radical probing (HRP) method, the orientation of RRF on the ribosome was derived. This study did not support the idea of direct molecular mimicry of tRNA by RRF, because the derived binding position of RRF was quite different from that one would expect based on structural mimicry. The inferred bin...

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Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 Å resolution by electron cryomicroscopy and angular reconstitution † 1 †This article is dedicated to the memory of Anneke van Heel. 1Edited by M.F. Moody

Cited by 200 publications

References 78 publications

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Domain organization of the type 1 inositol 1,4,5-trisphosphate receptor as revealed by single-particle analysis

Three-dimensional Model and Characterization of the Iron Stress-induced CP43′-Photosystem I Supercomplex Isolated from the Cyanobacterium Synechocystis PCC 6803

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

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