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1.
Mol Biol Cell ; 28(23): 3298-3314, 2017 Nov 07.
Article in English | MEDLINE | ID: mdl-28814505

ABSTRACT

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.


Subject(s)
Spindle Pole Bodies/metabolism , Spindle Pole Bodies/physiology , Bayes Theorem , Cell Cycle , Centrosome/metabolism , Computer Simulation , Crystallography, X-Ray/methods , Microtubule-Organizing Center/metabolism , Microtubules/metabolism , Mitosis , Nuclear Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Spindle Apparatus/metabolism , Structure-Activity Relationship , X-Ray Diffraction/methods
2.
Methods Mol Biol ; 857: 331-50, 2012.
Article in English | MEDLINE | ID: mdl-22323229

ABSTRACT

Advances in electron microscopy allow for structure determination of large biological machines at increasingly higher resolutions. A key step in this process is fitting component structures into the electron microscopy-derived density map of their assembly. Comparative modeling can contribute by providing atomic models of the components, via fold assignment, sequence-structure alignment, model building, and model assessment. All four stages of comparative modeling can also benefit from consideration of the density map. In this chapter, we describe numerous types of modeling problems restrained by a density map and available protocols for finding solutions. In particular, we provide detailed instructions for density map-guided modeling using the Integrative Modeling Platform (IMP), MODELLER, and UCSF Chimera.


Subject(s)
Macromolecular Substances/chemistry , Microscopy, Electron/methods , Models, Molecular , Proteins/chemistry , Amino Acid Sequence , Chaperonin 60/chemistry , Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Molecular Sequence Data , Protein Conformation , Sequence Alignment/methods
3.
J Biol Chem ; 286(1): 234-42, 2011 Jan 07.
Article in English | MEDLINE | ID: mdl-20962334

ABSTRACT

Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A- and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses.


Subject(s)
Bacteriophage T7/physiology , Capsid/chemistry , Capsid/metabolism , Bacteriophage T7/metabolism , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cryoelectron Microscopy , Models, Molecular , Protein Structure, Tertiary
4.
Curr Opin Cell Biol ; 21(1): 97-108, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19223165

ABSTRACT

Dynamic processes involving macromolecular complexes are essential to cell function. These processes take place over a wide variety of length scales from nanometers to micrometers, and over time scales from nanoseconds to minutes. As a result, information from a variety of different experimental and computational approaches is required. We review the relevant sources of information and introduce a framework for integrating the data to produce representations of dynamic processes.


Subject(s)
Cell Physiological Phenomena , Macromolecular Substances/metabolism , Computer Simulation , Models, Biological
5.
BMC Struct Biol ; 9: 6, 2009 Feb 17.
Article in English | MEDLINE | ID: mdl-19220918

ABSTRACT

BACKGROUND: It is well known the strong relationship between protein structure and flexibility, on one hand, and biological protein function, on the other hand. Technically, protein flexibility exploration is an essential task in many applications, such as protein structure prediction and modeling. In this contribution we have compared two different approaches to explore the flexibility space of protein domains: i) molecular dynamics (MD-space), and ii) the study of the structural changes within superfamily (SF-space). RESULTS: Our analysis indicates that the MD-space and the SF-space display a significant overlap, but are still different enough to be considered as complementary. The SF-space space is wider but less complex than the MD-space, irrespective of the number of members in the superfamily. Also, the SF-space does not sample all possibilities offered by the MD-space, but often introduces very large changes along just a few deformation modes, whose number tend to a plateau as the number of related folds in the superfamily increases. CONCLUSION: Theoretically, we obtained two conclusions. First, that function restricts the access to some flexibility patterns to evolution, as we observe that when a superfamily member changes to become another, the path does not completely overlap with the physical deformability. Second, that conformational changes from variation in a superfamily are larger and much simpler than those allowed by physical deformability. Methodologically, the conclusion is that both spaces studied are complementary, and have different size and complexity. We expect this fact to have application in fields as 3D-EM/X-ray hybrid models or ab initio protein folding.


Subject(s)
Computer Simulation , Protein Structure, Tertiary , Proteins/chemistry , Amino Acid Sequence , Models, Chemical , Models, Molecular , Sequence Analysis, Protein , Sequence Homology, Amino Acid
6.
Structure ; 15(4): 461-72, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17437718

ABSTRACT

The existence of similar folds among major structural subunits of viral capsids has shown unexpected evolutionary relationships suggesting common origins irrespective of the capsids' host life domain. Tailed bacteriophages are emerging as one such family, and we have studied the possible existence of the HK97-like fold in bacteriophage T7. The procapsid structure at approximately 10 A resolution was used to obtain a quasi-atomic model by fitting a homology model of the T7 capsid protein gp10 that was based on the atomic structure of the HK97 capsid protein. A number of fold similarities, such as the fitting of domains A and P into the L-shaped procapsid subunit, are evident between both viral systems. A different feature is related to the presence of the amino-terminal domain of gp10 found at the inner surface of the capsid that might play an important role in the interaction of capsid and scaffolding proteins.


Subject(s)
Bacteriophage T7/chemistry , Biological Evolution , Capsid/chemistry , Amino Acid Sequence , Bacteriophage T7/genetics , DNA , Molecular Sequence Data , Protein Binding , Protein Folding , Protein Structure, Tertiary
7.
J Virol ; 81(13): 6869-78, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17442720

ABSTRACT

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic alpha-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic alpha-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic alpha-helix in the precursor capsid, as a five-alpha-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.


Subject(s)
Capsid/chemistry , Infectious bursal disease virus/chemistry , Models, Molecular , Viral Structural Proteins/chemistry , Virus Assembly , Capsid/ultrastructure , Cryoelectron Microscopy , Infectious bursal disease virus/metabolism , Infectious bursal disease virus/ultrastructure , Nodaviridae/chemistry , Nodaviridae/ultrastructure , Protein Processing, Post-Translational , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Viral Structural Proteins/metabolism
8.
J Struct Biol ; 158(2): 165-81, 2007 May.
Article in English | MEDLINE | ID: mdl-17257856

ABSTRACT

We present a substantial improvement of S-flexfit, our recently proposed method for flexible fitting in three dimensional electron microscopy (3D-EM) at a resolution range of 8-12A, together with a comparison of the method capabilities with Normal Mode Analysis (NMA), application examples and a user's guide. S-flexfit uses the evolutionary information contained in protein domain databases like CATH, by means of the structural alignment of the elements of a protein superfamily. The added development is based on a recent extension of the Singular Value Decomposition (SVD) algorithm specifically designed for situations with missing data: Incremental Singular Value Decomposition (ISVD). ISVD can manage gaps and allows considering more aminoacids in the structural alignment of a superfamily, extending the range of application and producing better models for the fitting step of our methodology. Our previous SVD-based flexible fitting approach can only take into account positions with no gaps in the alignment, being appropriate when the superfamily members are relatively similar and there are few gaps. However, with new data coming from structural proteomics works, the later situation is becoming less likely, making ISVD the technique of choice for further works. We present the results of using ISVD in the process of flexible fitting with both simulated and experimental 3D-EM maps (GroEL and Poliovirus 135S cell entry intermediate).


Subject(s)
Algorithms , Imaging, Three-Dimensional/methods , Microscopy, Electron , Proteins/ultrastructure , Software , Chaperonin 60/ultrastructure , Mathematical Computing
9.
J Mol Biol ; 345(4): 759-71, 2005 Jan 28.
Article in English | MEDLINE | ID: mdl-15588824

ABSTRACT

In this paper the theoretical framework used to build a superfamily probability in electron microscopy (SPI-EM) is presented. SPI-EM is a new tool for determining the homologous superfamily to which a protein domain belongs looking at its three-dimensional electron microscopy map. The homologous superfamily is assigned according to the domain-architecture database CATH. Our method follows a probabilistic approach applied to the results of fitting protein domains into maps of proteins and the computation of local cross-correlation coefficient measures. The method has been tested and its usefulness proven with isolated domains at a resolution of 8 A and 12 A. Results obtained with simulated and experimental data at 10 A suggest that it is also feasible to detect the correct superfamily of the domains when dealing with electron microscopy maps containing multi-domain proteins. The inherent difficulties and limitations that multi-domain proteins impose are discussed. Our procedure is complementary to other techniques existing in the field to detect structural elements in electron microscopy maps like alpha-helices and beta-sheets. Based on the proposed methodology, a database of relevant distributions is being built to serve the community.


Subject(s)
Computer Simulation , Databases, Protein , Microscopy, Electron/methods , Proteins/classification , Proteins/ultrastructure , Color , Models, Molecular , Probability , Protein Structure, Tertiary , Proteins/chemistry , Software
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