2012Zhang IPET FETR: Difference between revisions
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== Citation == | == Citation == | ||
Zhang L, Ren G (2012) IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure. PLoS ONE 7(1): e30249 | |||
Zhang, | |||
== Abstract == | == Abstract == | ||
The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules. | The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules. | ||
== Keywords == | == Keywords == | ||
Protein dynamics; protein structure; equilibrium fluctuation; single-molecule structure; focused electron tomography reconstruction; FETR; individual-particle electron tomography; IPET; electron microscopy; EM; electron tomography; ET; cryo-electron tomography; CryoET. | |||
== Links == | == Links == | ||
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030249 | |||
https://www.nature.com/articles/s41598-020-66793-1 | |||
https://www.nature.com/articles/s41598-019-44978-7 | |||
https://pubmed.ncbi.nlm.nih.gov/30557627/ | |||
https://pubmed.ncbi.nlm.nih.gov/30420636/ | |||
https://www.nature.com/articles/s41467-018-03018-0 | |||
https://pubmed.ncbi.nlm.nih.gov/27621318/ | |||
https://pubmed.ncbi.nlm.nih.gov/27538822/ | |||
https://www.nature.com/articles/srep29231 | |||
https://www.nature.com/articles/srep09803 | |||
https://www.nature.com/articles/srep01089 | |||
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409128/ | |||
https://www.nature.com/articles/s41467-021-23966-4 | |||
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493104/ | |||
https://www.nature.com/articles/s41565-023-01321-6 | |||
== Related software == | == Related software == | ||
Latest revision as of 18:40, 24 August 2023
Citation
Zhang L, Ren G (2012) IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure. PLoS ONE 7(1): e30249
Abstract
The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.
Keywords
Protein dynamics; protein structure; equilibrium fluctuation; single-molecule structure; focused electron tomography reconstruction; FETR; individual-particle electron tomography; IPET; electron microscopy; EM; electron tomography; ET; cryo-electron tomography; CryoET.
Links
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030249
https://www.nature.com/articles/s41598-020-66793-1
https://www.nature.com/articles/s41598-019-44978-7
https://pubmed.ncbi.nlm.nih.gov/30557627/
https://pubmed.ncbi.nlm.nih.gov/30420636/
https://www.nature.com/articles/s41467-018-03018-0
https://pubmed.ncbi.nlm.nih.gov/27621318/
https://pubmed.ncbi.nlm.nih.gov/27538822/
https://www.nature.com/articles/srep29231
https://www.nature.com/articles/srep09803
https://www.nature.com/articles/srep01089
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409128/
https://www.nature.com/articles/s41467-021-23966-4
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493104/
https://www.nature.com/articles/s41565-023-01321-6