Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

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Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

10.1101/2021.08.09.455715 ◽

2021 ◽

Author(s):

Xudong Wu ◽

Tom A Rapoport

Keyword(s):

Structure Determination ◽

Model Building ◽

De Novo ◽

Binding Protein ◽

Protein A ◽

Fab Fragment ◽

Binding Domains ◽

Binding Interface ◽

Small Proteins

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: (1) a Fab-fragment of an antibody directed against the nanobody, and (2) a nanobody-binding protein A fragment fused to maltose-binding protein and Fab-binding domains. The overall ensemble of ~120 kDa, called Legobody, does not perturb the nanobody-target interaction and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23 kDa membrane protein, resulting in a map at 3.2 Angstrom overall resolution with density sufficient for de novo model building, and for the 22 kDa RBD of SARS-CoV2 spike protein, resulting in a map at 3.6 Angstrom overall resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

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Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module 1 1Edited by D. C. Rees

Journal of Molecular Biology ◽

10.1006/jmbi.1999.3398 ◽

2000 ◽

Vol 295 (3) ◽

pp. 541-556 ◽

Cited By ~ 88

Author(s):

Adrian R. Ferré-D’Amaré ◽

Jennifer A. Doudna

Keyword(s):

Structure Determination ◽

Hepatitis Delta Virus ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Hepatitis Delta ◽

Hepatitis Delta Virus Ribozyme ◽

Delta Virus

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An Approach for De Novo Structure Determination of Dynamic Molecular Assemblies by Electron Cryomicroscopy

Structure ◽

10.1016/j.str.2010.05.001 ◽

2010 ◽

Vol 18 (6) ◽

pp. 667-676 ◽

Cited By ~ 21

Author(s):

Bjoern Sander ◽

Monika M. Golas ◽

Reinhard Lührmann ◽

Holger Stark

Keyword(s):

Structure Determination ◽

De Novo ◽

Electron Cryomicroscopy ◽

Molecular Assemblies

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Proton and nitrogen sequential assignments and secondary structure determination of the human FK506 and rapamycin binding protein

Biochemistry ◽

10.1021/bi00233a020 ◽

1991 ◽

Vol 30 (19) ◽

pp. 4774-4789 ◽

Cited By ~ 30

Author(s):

M. K. Rosen ◽

S. W. Michnick ◽

M. Karplus ◽

S. L. Schreiber

Keyword(s):

Secondary Structure ◽

Structure Determination ◽

Binding Protein

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Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera

International Journal of Molecular Sciences ◽

10.3390/ijms22136859 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6859

Author(s):

Chun-Hsiung Wang ◽

Dong-Hua Chen ◽

Shih-Hsin Huang ◽

Yi-Min Wu ◽

Yi-Yun Chen ◽

...

Keyword(s):

High Resolution ◽

Structure Determination ◽

De Novo ◽

Macrobrachium Rosenbergii ◽

Test Sample ◽

Depth Of Field ◽

Necrosis Virus ◽

Electron Counting ◽

Global Pandemic

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes. Here, we investigated the capability of 200 kV multipurpose microscopes equipped with a direct electron camera in determining the structures of infectious particles. We used 30 nm particles of the grouper nerve necrosis virus as a test sample and obtained the cryo-EM structure with a resolution as high as ∼2.7 Å from a setting that used electron counting. For comparison, we tested a high-end cryo-EM (Talos Arctica) using a similar virus (Macrobrachium rosenbergii nodavirus) to obtain virtually the same resolution. Those results revealed that the resolution is ultimately limited by the depth of field. Our work updates the density maps of these viruses at the sub-3Å level to allow for building accurate atomic models from de novo to provide structural insights into the assembly of the capsids. Importantly, this study demonstrated that multipurpose TEMs are capable of the high-resolution cryo-EM structure determination of infectious particles and is thus germane to the research on pandemics.

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De novo structure determination of butadiene by isotope-resolved rotational Raman spectroscopy

Physical Chemistry Chemical Physics ◽

10.1039/d0cp00129e ◽

2020 ◽

Vol 22 (16) ◽

pp. 8933-8939

Author(s):

Begüm Rukiye Özer ◽

In Heo ◽

Jong Chan Lee ◽

Christian Schröter ◽

Thomas Schultz

Keyword(s):

Molecular Structure ◽

Raman Spectroscopy ◽

Structure Determination ◽

De Novo ◽

Rotational Spectroscopy ◽

Theoretical Predictions

Isotope-selective rotational spectroscopy allows determination of molecular structure independent of assumptions or theoretical predictions.

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A fragmentation and reassembly method forab initiophasing

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714025449 ◽

2015 ◽

Vol 71 (2) ◽

pp. 304-312 ◽

Cited By ~ 14

Author(s):

Rojan Shrestha ◽

Kam Y. J. Zhang

Keyword(s):

Ab Initio ◽

Structure Determination ◽

Model Building ◽

De Novo ◽

Sequence Information ◽

Molecular Replacement ◽

Protein Targets ◽

Current State ◽

Viable Approach ◽

Automated Model Building

Ab initiophasing withde novomodels has become a viable approach for structural solution from protein crystallographic diffraction data. This approach takes advantage of the known protein sequence information, predictsde novomodels and uses them for structure determination by molecular replacement. However, even the current state-of-the-artde novomodelling method has a limit as to the accuracy of the model predicted, which is sometimes insufficient to be used as a template for successful molecular replacement. A fragment-assembly phasing method has been developed that starts from an ensemble of low-accuracyde novomodels, disassembles them into fragments, places them independently in the crystallographic unit cell by molecular replacement and then reassembles them into a whole structure that can provide sufficient phase information to enable complete structure determination by automated model building. Tests on ten protein targets showed that the method could solve structures for eight of these targets, although the predictedde novomodels cannot be used as templates for successful molecular replacement since the best model for each target is on average more than 4.0 Å away from the native structure. The method has extended the applicability of theab initiophasing byde novomodels approach. The method can be used to solve structures when the bestde novomodels are still of low accuracy.

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Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606287113 ◽

2016 ◽

Vol 113 (40) ◽

pp. 11232-11236 ◽

Cited By ~ 50

Author(s):

Michael R. Sawaya ◽

Jose Rodriguez ◽

Duilio Cascio ◽

Michael J. Collazo ◽

Dan Shi ◽

...

Keyword(s):

Ab Initio ◽

Structure Determination ◽

De Novo ◽

Direct Methods ◽

Biological Macromolecules ◽

X Ray Diffraction ◽

Em Method ◽

Ab Initio Structure ◽

Diffraction Patterns

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

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