Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

Atomic-resolution structure determination is the key requirement for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enabled determination of the high-resolution structure of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Angstrom by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and specifically labelled methyl groups, and a 4.1 Angstrom resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution (up to 8 Angstrom) cryo-EM data are available, thus paving avenues for the structure determination of challenging biological assemblies.

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Towards Atomic-Resolution Structure Determination of HIV-1 Capsid Assemblies using Magic Angle Spinning NMR

Biophysical Journal ◽

10.1016/j.bpj.2018.11.1682 ◽

2019 ◽

Vol 116 (3) ◽

pp. 310a

Author(s):

Manman Lu ◽

Mingzhang Wang ◽

Jochem Struppe ◽

Werner Maas ◽

Angela Gronenborn ◽

...

Keyword(s):

Structure Determination ◽

Magic Angle Spinning ◽

Atomic Resolution ◽

Magic Angle ◽

Angle Spinning ◽

Hiv 1 ◽

Resolution Structure

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Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

Journal of the American Chemical Society ◽

10.1021/ja4021149 ◽

2013 ◽

Vol 135 (22) ◽

pp. 8294-8303 ◽

Cited By ~ 35

Author(s):

Jianping Li ◽

Kala Bharath Pilla ◽

Qingfeng Li ◽

Zhengfeng Zhang ◽

Xuncheng Su ◽

...

Keyword(s):

Structure Determination ◽

Magic Angle Spinning ◽

Nmr Structure ◽

Magic Angle ◽

Pseudocontact Shifts ◽

Nmr Structure Determination ◽

Angle Spinning

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A resolution record for cryoEM

Faculty Reviews ◽

10.12703/r-01-000002 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jonathan Ashmore ◽

Bridget Carragher ◽

Peter B Rosenthal ◽

William Weis

Keyword(s):

Electron Microscopy ◽

Image Analysis ◽

Structure Determination ◽

Test Specimen ◽

Structural Information ◽

Atomic Resolution ◽

Fast Growing ◽

Cryo Electron Microscopy ◽

New Instrument ◽

Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

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Structural Properties of a Dimeric Phenylcyanamidocopper(I) Complex, [{Cu(PPh3)2(C6H5NCN)}2]

Australian Journal of Chemistry ◽

10.1071/ch00145 ◽

2000 ◽

Vol 53 (12) ◽

pp. 971 ◽

Cited By ~ 5

Author(s):

Eric W. Ainscough ◽

Andrew M. Brodie ◽

Peter C. Healy ◽

Joyce M. Waters

Keyword(s):

Crystal Structure ◽

Solid State ◽

Structural Properties ◽

Structure Determination ◽

Magic Angle Spinning ◽

Magic Angle ◽

Cross Polarization ◽

X Ray ◽

Angle Spinning

The X-ray crystal structure determination of bis[-(phenylcyanamido)bis(triphenylphosphine)copper(I)], [{Cu(PPh3)2(C6H5NCN)}2], (1) is reported. The complex has a centrosymmetric dimeric structure with the phenylcyanamide ligands bridging the copper atoms in a -1,3-fashion. The structure is compared with that of the 4-methylphenylcyanamido complex, [{Cu(PPh3)2(4-MeC6H4NCN)}2] (2), and the differences observed in the Cu–P bond lengths compared with changes in the solid state 31P cross-polarization magic-angle spinning (CPMAS) spectra of the two complexes.

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Dihedral Angle Measurements for Structure Determination by Biomolecular Solid-State NMR Spectroscopy

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.791090 ◽

2021 ◽

Vol 8 ◽

Author(s):

Patrick C. A. van der Wel

Keyword(s):

Solid State ◽

Structure Determination ◽

Solid State Nmr ◽

Structural Information ◽

Magic Angle Spinning ◽

Magic Angle ◽

Distance Measurements ◽

Challenges And Opportunities ◽

Molecule Interactions ◽

Angle Spinning

In structural studies of immobilized, aggregated and self-assembled biomolecules, solid-state NMR (ssNMR) spectroscopy can provide valuable high-resolution structural information. Among the structural restraints provided by magic angle spinning (MAS) ssNMR the canonical focus is on inter-atomic distance measurements. In the current review, we examine the utility of ssNMR measurements of angular constraints, as a complement to distance-based structure determination. The focus is on direct measurements of angular restraints via the judicious recoupling of multiple anisotropic ssNMR parameters, such as dipolar couplings and chemical shift anisotropies. Recent applications are highlighted, with a focus on studies of nanocrystalline polypeptides, aggregated peptides and proteins, receptor-substrate interactions, and small molecule interactions with amyloid protein fibrils. The review also examines considerations of when and where ssNMR torsion angle experiments are (most) effective, and discusses challenges and opportunities for future applications.

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Atomic-resolution structure of HIV-1 capsid tubes by magic-angle spinning NMR

Nature Structural & Molecular Biology ◽

10.1038/s41594-020-0489-2 ◽

2020 ◽

Vol 27 (9) ◽

pp. 863-869 ◽

Cited By ~ 8

Author(s):

Manman Lu ◽

Ryan W. Russell ◽

Alexander J. Bryer ◽

Caitlin M. Quinn ◽

Guangjin Hou ◽

...

Keyword(s):

Magic Angle Spinning ◽

Atomic Resolution ◽

Magic Angle ◽

Angle Spinning ◽

Hiv 1 ◽

Resolution Structure

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Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex

Nature Communications ◽

10.1038/s41467-019-10490-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 27

Author(s):

Diego F. Gauto ◽

Leandro F. Estrozi ◽

Charles D. Schwieters ◽

Gregory Effantin ◽

Pavel Macek ◽

...

Keyword(s):

Structure Determination ◽

Atomic Resolution ◽

Enzyme Complex ◽

Resolution Structure

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Structure Determination in Combinatorial Chemistry: Utilization of Magic Angle Spinning HMQC and TOCSY NMR Spectra in the Structure Determination of Wang-bound Lysine.

Tetrahedron Letters ◽

10.1016/00404-0399(50)1022a- ◽

1995 ◽

Vol 36 (30) ◽

pp. 5311-5314

Author(s):

R Anderson

Keyword(s):

Combinatorial Chemistry ◽

Structure Determination ◽

Nmr Spectra ◽

Magic Angle Spinning ◽

Magic Angle ◽

Angle Spinning

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Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1509852112 ◽

2015 ◽

Vol 112 (47) ◽

pp. 14611-14616 ◽

Cited By ~ 30

Author(s):

Si Yan ◽

Changmiao Guo ◽

Guangjin Hou ◽

Huilan Zhang ◽

Xingyu Lu ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Cell Biology ◽

Double Resonance ◽

Magic Angle Spinning ◽

Atomic Resolution ◽

Organelle Transport ◽

Magic Angle ◽

Associated Proteins ◽

Angle Spinning ◽

Resolution Structure

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

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