Movies of horse spleen apoferritin on multifunctional ultrastable graphene supports for electron cryomicroscopy
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ABSTRACT:
SUBMITTER: Christopher John Russo
PROVIDER: EMPIAR-10272 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:With recent technological advances, the atomic resolution structure of any purified biomolecular complex can, in principle, be determined by single-particle electron cryomicroscopy (cryoEM). In practice, the primary barrier to structure determination is the preparation of a frozen specimen suitable for high-resolution imaging. To address this, we present a multifunctional specimen support for cryoEM, comprising large-crystal monolayer graphene suspended across the surface of an ultrastable gold specimen support. Using a low-energy plasma surface modification system, we tune the surface of this support to the specimen by patterning a range of covalent functionalizations across the graphene layer on a single grid. This support design reduces specimen movement during imaging, improves image quality, and allows high-resolution structure determination with a minimum of material and data.
| S-EPMC6575631 | biostudies-literature
Project description: Not available
| EMPIAR-10026 | biostudies-other
Project description:1. The dissociation of horse spleen apoferritin as a function of pH was analysed by sedimentation-velocity techniques. The oligomer is stable in the range pH2.8-10.6. Between pH2.8 and 1.6 and 10.6 and 13.0 both oligomer and subunits can be detected. At pH values between 1.6 and 1.0 the subunit is the only species observed, although below pH1.0 aggregation of the subunits to a particle sedimenting much faster than the oligomer occurs. 2. When apoferritin is first dissociated into subunits at low pH values and then dialysed into buffers of pH1.5-5.0, the subunit reassociates to oligomer in the pH range 3.1-4.3. 3. U.v.-difference spectroscopy was used to study conformational changes occurring during the dissociation process. The difference spectrum in acid can be accounted for by the transfer of four to five tyrosine residues/subunit from the interior of the protein into the solvent. This process is reversed on reassociation, but shows the same hysteresis as found by sedimentation techniques. The difference spectrum in alkali is more complex, but is consistent with the deprotonation of tyrosine residues, which appear to have rather high pK values. 4. In addition to the involvement of tyrosine residues in the conformational change at low pH values, spectral evidence is presented that one tryptophan residue/subunit also changes its environment before dissociation and subsequent to reassociation. 5. Analysis of the dissociation and reassociation of apoferritin at low pH values suggests that this is a co-operative process involving protonation and deprotonation of at least two carboxyl functions of rather low intrinsic pK. The dissociation at alkaline pH values does not appear to be co-operative. 6. Of the five tyrosine residues/subunit only one can be nitrated with tetranitromethane. Guanidination of lysine residues results in the modification of seven out of a total of nine residues/subunit. Nine out of the ten arginine residues/subunit react with cyclohexanedione.
| S-EPMC1177698 | biostudies-other
Project description:Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ?490 kDa protein complex ?20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.
| S-EPMC5647560 | biostudies-literature
Project description:1. Horse spleen apoferritin catalyses the oxidation of Fe(2+) to Fe(3+) with molecular O(2) as electron acceptor under conditions where a number of other proteins have no such effect. The product is similar to ferritin by a number of criteria. 2. The progress curve is hyperbolic and the increase in initial velocity is linear with increasing apoferritin concentration. With respect to Fe(2+) the reaction follows Michaelis-Menten kinetics. The pH-dependence of the reaction was determined between pH4.3 and 6.0. 3. Modification of both tryptophan residues/apoferritin subunit with 2-nitrophenylsulphenyl chloride does not affect either k(cat.) or K(m) for the oxidation. Neither does the guanidination of seven out of nine lysine residues/subunit, the modification of nine out of ten arginine residues/subunit with cyclohexanedione, or the nitration of one out of five tyrosine residues/subunit with tetranitromethane. 4. The carboxymethylation of two out of three cysteine residues/subunit and of one out of six histidine residues/subunit can be achieved with iodoacetic acid. This carboxymethylated apoferritin is completely inactive in Fe(2+) oxidation. 5. Apoferritin does not take up Fe(3+). It appears from these results that Fe(2+) is the form in which iron is taken up by ferritin in a reaction where the protein acts as an enzyme which traps the product in the interior of the protein shell.
| S-EPMC1177699 | biostudies-other
Project description:Despite recent advances, the structures of many proteins cannot be determined by electron cryomicroscopy because the individual proteins move during irradiation. This blurs the images so that they cannot be aligned with each other to calculate a three-dimensional density. Much of this movement stems from instabilities in the carbon substrates used to support frozen samples in the microscope. Here we demonstrate a gold specimen support that nearly eliminates substrate motion during irradiation. This increases the subnanometer image contrast such that α helices of individual proteins are resolved. With this improvement, we determine the structure of apoferritin, a smooth octahedral shell of α-helical subunits that is particularly difficult to solve by electron microscopy. This advance in substrate design will enable the solution of currently intractable protein structures.
| S-EPMC4296556 | biostudies-literature
Project description: Not available
| EMPIAR-10474 | biostudies-other
Project description: Not available
| EMPIAR-11254 | biostudies-other
Project description: Not available
| EMPIAR-11260 | biostudies-other
Project description: Not available
| EMPIAR-11261 | biostudies-other