Dong Hua Chen

De Novo Backbone Trace of GroEL from Single Particle Electron Cryomicroscopy.

In this work, we employ single-particle electron cryo-microscopy (cryo-EM) to reconstruct GroEL to approximately 4 A resolution with both D7 and C7 symmetry. Using a newly developed skeletonization algorithm and secondary structure element identification in combination with sequence-based secondary structure prediction, we demonstrate that it is possible to achieve a de novo Calpha trace directly from a cryo-EM reconstruction. The topology of our backbone trace is completely accurate, though subtle alterations illustrate significant differences from existing crystal structures. In the map with C7 symmetry, the seven monomers in each ring are identical; however, the subunits have a subtly different structure in each ring, particularly in the equatorial domain. These differences include an asymmetric salt bridge, density in the nucleotide-binding pocket of only one ring, and small shifts in alpha helix positions. This asymmetric conformation is different from previous asymmetric structures, including GroES-bound GroEL, and may represent a primed state in the chaperonin pathway.

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

An atomic model of brome mosaic virus using direct electron detection and real-space optimization

Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8u2009Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

The Pattern of Tegument-Capsid Interaction in the Herpes Simplex Virus Type 1 Virion Is Not Influenced by the Small Hexon-Associated Protein VP26

ABSTRACT Examination of the three-dimensional structure of intact herpes simplex virus type 1 (HSV-1) virions had revealed that the icosahedrally symmetrical interaction between the tegument and capsid involves the pentons but not the hexons (Z. H. Zhou, D. H. Chen, J. Jakana, F. J. Rixon, and W. Chiu, J. Virol. 73:3210–3218, 1999). To account for this, we postulated that the presence of the small capsid protein, VP26, on top of the hexons was masking potential binding sites and preventing tegument attachment. We have now tested this hypothesis by determining the structure of virions lacking VP26. Apart from the obvious absence of VP26 from the capsids, the structures of the VP26 minus and wild-type virions were essentially identical. Notably, they showed the same tegument attachment patterns, thereby demonstrating that VP26 is not responsible for the divergent tegument binding properties of pentons and hexons.

Achievable resolution from images of biological specimens acquired from a 4k × 4k CCD camera in a 300-kV electron cryomicroscope

Bacteriorhodopsin and epsilon 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k x 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000x effective magnification averaged 15.7 degrees out to 5.8-A resolution relative to Hendersons published values. Using a single-particle reconstruction technique, we obtained an 8.2-A icosahedral structure of epsilon 15 bacteriophage with the CCD images collected at an effective magnification of 56,000x. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.

GroEL Structure at 6 Å Resolution Using Electron Cryomicroscopy and EMAN

We present a reconstruction of native apo-GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at ~6 A resolution.uf020This continues the long history of cryo-EM contributions to GroEL research, for example [1,2,3]. As this was largely a test of the reconstruction methodology, and we wished to avoid any possibility of biasing the results, absolutely no reference was made to the x-ray crystal structure [4] at any point during the actual reconstruction process. To perform this reconstruction, 39,085 particles were selected from 42 micrographs collected on a JEOL 2010F electron cryomicroscope. The resolution achieved in this reconstruction was largely due to new features added to the EMAN [5] software suite. While the overall reconstruction methodology remains unchanged, substantial improvements were made to several specific algorithms. Through improvements to the 2-D particle alignment routines, use of improved similarity criteria in particle classification, and reductions in iterative particle class-alignment, we were able to achieve the presented structure at ~6 A resolution as measured by Fourier Shell Correlation using the 0.5 criterion.