Ab initio resolution measurement for single particle structures

Sousa, Duncan; Grigorieff, Nikolaus

doi:10.1016/j.jsb.2006.08.003

Cited by 101 publications

(99 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Rmeasure was even less reliable as it tends to fail with low resolution structures (Sousa and Grigorieff, 2007). We saw a sharp break in the distribution of resolutions we obtained for the initial models with Rmeasure, with about half of our reconstructions in the 35Å to 47Å range and the rest all giving a resolution of 90Å (data not shown).…”

Section: How Do We Select the Best Initial Models From A Large Set Ofmentioning

confidence: 86%

“…We tested both the Fourier Shell Correlation (FSC) (Harauz and van Heel, 1986) and Rmeasure (Sousa and Grigorieff, 2007). Unfortunately, while a few of the "good" volumes shown in Figure 2A had relatively good resolutions by the 0.5 FSC criterion, others ranked near the bottom of our 101 initial volumes.…”

Section: How Do We Select the Best Initial Models From A Large Set Ofmentioning

confidence: 99%

See 1 more Smart Citation

Validation of the orthogonal tilt reconstruction method with a biological test sample

Chandramouli

Hernández-López

Wang

et al. 2011

Journal of Structural Biology

View full text Add to dashboard Cite

Section: How Do We Select the Best Initial Models From A Large Set Ofmentioning

confidence: 86%

Section: How Do We Select the Best Initial Models From A Large Set Ofmentioning

confidence: 99%

Validation of the orthogonal tilt reconstruction method with a biological test sample

Chandramouli

Hernández-López

Wang

et al. 2011

Journal of Structural Biology

View full text Add to dashboard Cite

“…These particles were then subjected to 10 subsequent rounds of refinement while enforcing either C1 or I1 symmetry, and while dividing the particles into two equal halves. The resulting 3D structures were resolved to ∼15 Å as independently verified using the RMEASURE program [12]. Overall parameters for the global reconstruction routines included a pixel size of 5 Å, a reference model low-pass filtered to 50 Å, and a regularization parameter of T=4 over an angular search space of 7.5 degrees.…”

Section: Image Processing and 3d Refinementmentioning

confidence: 99%

A Non-Symmetric Reconstruction Technique for Transcriptionally-Active Viral Assemblies

Rahimi

Varano

Demmert

et al. 2015

J Analyt Molecul Tech

View full text Add to dashboard Cite

The molecular mechanisms by which RNA viruses coordinate their transcriptional activities are not fully understood. For rotavirus, an important pediatric gastroenteric pathogen, transcription occurs within a double-layered particle that encloses the viral genome. To date, there remains very little structural information available for actively-transcribing rotavirus double-layered particles, which could provide new insights for antiviral development. To improve our vision of these viral assemblies, we developed a new combinatorial strategy that utilizes currently available highresolution image processing tools. First, we employed a 3D classification routine that allowed us to sort transcriptionally-active rotavirus assemblies on the basis of their internal density. Next, we implemented an additional 3D refinement procedure using the most active class of DLPs. For comparison, the refined structures were computed in parallel by (1) enforcing icosahedral symmetry, and by (2) using no symmetry operators. Comparing the resulting structures, we were able to visualize the continuum that exists between viral capsid proteins and the viral RNA for the first time.

show abstract

“…EM reconstructions were calculated for each type of specimen and included 572 particles for images of DLPs over holes ( Figure 3E) and 38 particles for images of DLPs on cryo-SiN (Figure 3F). Using the RMEASURE program [16] the reconstruction calculated for DLPs in holes was approximately 2.4 nm while the reconstruction calculated for DLPs on cryo-SiN was estimated to be 2.5 nm. As a control, we selected 40 random particles from specimens prepared on holey carbon film and used the same computing parameters.…”

Section: D Reconstructions Reveal Conserved Capsids With Variable Coresmentioning

confidence: 99%

Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

2013

J Analyt Molecul Tech

View full text Add to dashboard Cite

Silicon nitride (SiN) membranes that are merely nanometers-thick have been introduced as new support films for electron microscopy (EM) applications. The use of these thin membranes for high-resolution imaging is widely attractive due to their consistent manufacture, flatness, and durability. Here, we utilized SiN support films to actively monitor key steps in rotavirus RNA synthesis at the nanoscale. Rotaviruses are gastrointestinal pathogens that infect pediatric populations and pose a major health problem in developing countries. We demonstrate, for the first time, that cryo-EM specimens of these viral assemblies may be prepared using SiN membranes, that we have termed "cryo-SiN". Cryo-SiN microchips containing inactive and actively transcribing rotavirus complexes were vitrified using gentleblot conditions to yield reproducibly thin ice-embedded specimens. EM imaging results indicate that cryo-SiN samples exhibited greater visual contrast than specimens prepared on conventional support films under the same conditions. The improved contrast of the cryo-SiN samples permitted us to calculate 3D structures of rotavirus complexes using a fraction of the images required for conventionally prepared specimens. We anticipate the use of cryo-SiN microchip will enable us to better investigate critical viral life-cycle processes at the molecular level under near-native conditions. AbbreviationsDouble-Layered Particles (DLPs); Silicon Nitride (SiN); Electron Microscopy (EM); Transmission Electron Microscope (TEM); Nickel-Nitrilotriacetic Acid (Ni-NTA); Nanometers (nm)

show abstract

Ab initio resolution measurement for single particle structures

Cited by 101 publications

References 27 publications

Validation of the orthogonal tilt reconstruction method with a biological test sample

Validation of the orthogonal tilt reconstruction method with a biological test sample

A Non-Symmetric Reconstruction Technique for Transcriptionally-Active Viral Assemblies

Cryo-SiN – An Alternative Substrate to Visualize Active Viral Assemblies

Contact Info

Product

Resources

About