scholarly journals Large protein complex production using the SmartBac System - Strategies and Applications

2017 ◽  
Author(s):  
Yujia Zhai ◽  
Danyang Zhang ◽  
Leiye Yu ◽  
Fang Sun ◽  
Fei Sun
Keyword(s):  
Structural Biology ◽  
Insect Cells ◽  
Fluorescent Proteins ◽  
Large Protein ◽  
Gibson Assembly ◽  
Multiprotein Complexes ◽  
Assembly Method ◽  
Cryo Electron Microscopy ◽  
Large Protein Complex ◽  
Dynactin Complex

ABSTRACTRecent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and versatile system for constructing large-sized transfer plasmids used to generate recombinant baculoviruses that express large multiprotein complexes in insect cells. The SmartBac system integrates the univector plasmid-fusion system, Gibson assembly method and polyprotein strategy to construct the final transfer plasmids. The fluorescent proteins are designed to be co-expressed with recombinant proteins to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for effective purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed, suggesting a great potential of SmartBac for its wide application in the future structural biology study.

scholarly journals Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method

2015 ◽  
Vol 81 (20) ◽  
pp. 7244-7252 ◽  
Author(s):  
Katrin Beilharz ◽  
Renske van Raaphorst ◽  
Morten Kjos ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Gibson Assembly ◽  
Content Type ◽  
Assembly Method ◽  
Red Fluorescent Proteins ◽  
Wide Range

ABSTRACTDuring the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogenStreptococcus pneumoniaeand other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) inS. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP inS. pneumoniaeand is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling inS. pneumoniaeand related organisms.

scholarly journals Structure of the human inner kinetochore CCAN complex and its significance for human centromere organization

2022 ◽  
Author(s):  
Marion E. Pesenti ◽  
Tobias Raisch ◽  
Duccio Conti ◽  
Ingrid Hoffmann ◽  
Dorothee Vogt ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Chromosome Segregation ◽  
Histone H3 ◽  
Large Protein ◽  
Centromeric Chromatin ◽  
Human Centromere ◽  
Cryo Electron Microscopy ◽  
Large Protein Complex ◽  
Histone H3 Variant ◽  
Specificity Determinants

Centromeres are specialized chromosome loci that seed the kinetochore, a large protein complex that effects chromosome segregation. The organization of the interface between the kinetochore and the specialized centromeric chromatin, marked by the histone H3 variant CENP-A, remains incompletely understood. A 16-subunit complex, the constitutive centromere associated network (CCAN), bridges CENP-A to the spindle-binding moiety of the kinetochore. Here, we report a cryo-electron microscopy structure of human CCAN. We highlight unique features such as the pseudo GTPase CENP-M and report how a crucial CENP-C motif binds the CENP-LN complex. The CCAN structure has also important implications for the mechanism of specific recognition of the CENP-A nucleosome. A supported model depicts the interaction as fuzzy and identifies the disordered CCAN subunit CENP-C as only determinant of specificity. A more speculative model identifies both CENP-C and CENP-N as specificity determinants, but implies CENP-A may be in a hemisome rather than in a classical octamer.

Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽  
2021 ◽  
Author(s):  
Edward Egelman ◽  
Fengbin Wang
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Macromolecular Complexes ◽  
Atomic Structures ◽  
Cryo Electron Microscopy ◽  
Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

scholarly journals Single-particle cryo-EM—How did it get here and where will it go

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. 876-880 ◽  
Author(s):  
Yifan Cheng
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Single Particle ◽  
Electron Crystallography ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Technological Advances ◽  
The Future ◽  
Electron Cryotomography

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

scholarly journals Reconstitution of the recombinant human γ-tubulin ring complex

Open Biology ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 200325
Author(s):  
Martin Würtz ◽  
Anna Böhler ◽  
Annett Neuner ◽  
Erik Zupa ◽  
Lukas Rohland ◽  
...  
Keyword(s):  
Insect Cells ◽  
Recombinant Expression ◽  
Expression System ◽  
Cryo Electron Microscopy ◽  
Egg Extract ◽  
Activation Mechanisms ◽  
Ring Complex ◽  
Core Components ◽  
End Capping ◽  
Recombinant Expression System

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

scholarly journals Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

2021 ◽  
Author(s):  
Nicole Dimos ◽  
Carl P.O. Helmer ◽  
Andrea M. Chanique ◽  
Markus C. Wahl ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Structure Analysis ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Fast Development ◽  
Pharmaceutical Industries ◽  
Exquisite Specificity ◽  
Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

Construction of full-length infectious cDNA clones of Apple stem grooving virus using Gibson Assembly method

2020 ◽  
Vol 276 ◽  
pp. 197790
Author(s):  
Zheng-Nan Li ◽  
Wilhelm Jelkmann ◽  
Ping-ping Sun ◽  
Lei Zhang
Keyword(s):  
Full Length ◽  
Cdna Clones ◽  
Gibson Assembly ◽  
Assembly Method ◽  
Apple Stem Grooving Virus ◽  
Apple Stem

Uncovering the structures of modular polyketide synthases

2015 ◽  
Vol 32 (3) ◽  
pp. 436-453 ◽  
Author(s):  
Kira J. Weissman
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Structural Characterization ◽  
Small Angle ◽  
Single Particle ◽  
Polyketide Synthases ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

This review covers a breakthrough in the structural biology of the gigantic modular polyketide synthases (PKS): the structural characterization of intact modules by single-particle cryo-electron microscopy and small-angle X-ray scattering.

Sign in / Sign up

Export Citation Format

Share Document