Direct structure determination by atomic-resolution incoherent STEM imaging

Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.

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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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The Role of Validation in Macromolecular Crystallography

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444998007136 ◽

1998 ◽

Vol 54 (6) ◽

pp. 1109-1118 ◽

Cited By ~ 2

Author(s):

Eleanor Dodson

Keyword(s):

Structure Determination ◽

Atomic Resolution ◽

File Format ◽

Macromolecular Crystallography ◽

X Ray

The importance of validation techniques in X-ray structure determination and their relation to refinement procedures are discussed, with particular reference to atomic resolution structures. The requirements of deposition and publication, and the role of validation tools in this are analysed. The need for a rigorously defined file format is emphasized.

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Organic structure determination using atomic-resolution scanning probe microscopy

Nature Chemistry ◽

10.1038/nchem.765 ◽

2010 ◽

Vol 2 (10) ◽

pp. 821-825 ◽

Cited By ~ 236

Author(s):

Leo Gross ◽

Fabian Mohn ◽

Nikolaj Moll ◽

Gerhard Meyer ◽

Rainer Ebel ◽

...

Keyword(s):

Structure Determination ◽

Scanning Probe Microscopy ◽

Scanning Probe ◽

Atomic Resolution ◽

Probe Microscopy ◽

Organic Structure

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Structure determination from X-ray powder diffraction data at low resolution

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314098568 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C143-C143

Author(s):

Hongliang Xu

Keyword(s):

Single Crystal ◽

Diffraction Pattern ◽

Crystal Structures ◽

Powder Diffraction ◽

Diffraction Data ◽

Structure Determination ◽

Direct Methods ◽

Atomic Resolution ◽

Powder Diffraction Data ◽

Low Resolution

Knowledge of the structural arrangement of atoms in solids is necessary to facilitate the study of their properties. The best and most detailed structural information is obtained when the diffraction pattern of a single crystal a few tenths of a millimeter in each dimension is analyzed, but growing high-quality crystals of this size is often difficult, sometimes impossible. However, many crystallization experiments that do not yield single crystals do yield showers of randomly oriented micro-crystals that can be exposed to X-rays simultaneously to produce a powder diffraction pattern. Direct Methods routinely solve crystal structures when single-crystal diffraction data are available at atomic resolution (1.0-1.2Å), but fail to determine micro-crystal structures due to reflections overlapping and low-resolution powder diffraction data. By artificially and intelligently extending the measured data to atomic resolution, we have successfully solved structures having low-resolution diffraction data that were hard to solve by other direct-method based computation procedures. The newly developed method, Powder Shake-and-Bake, is implemented in a computer program PowSnB. PowSnB can be incorporated into the state-of-the-art software package EXPO that includes powder data reduction, structure determination and structure refinement. The new combination could have potential to solve structures that have never been solved before by direct-methods approach.

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De novo protein structure determination from near-atomic-resolution cryo-EM maps

Nature Methods ◽

10.1038/nmeth.3287 ◽

2015 ◽

Vol 12 (4) ◽

pp. 335-338 ◽

Cited By ~ 119

Author(s):

Ray Yu-Ruei Wang ◽

Mikhail Kudryashev ◽

Xueming Li ◽

Edward H Egelman ◽

Marek Basler ◽

...

Keyword(s):

Protein Structure ◽

Structure Determination ◽

De Novo ◽

Protein Structure Determination ◽

Atomic Resolution

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

10.1101/498287 ◽

2018 ◽

Author(s):

Diego Gauto ◽

Leandro Estrozi ◽

Charles Schwieters ◽

Gregory Effantin ◽

Pavel Macek ◽

...

Keyword(s):

Structure Determination ◽

Protein Function ◽

Structural Information ◽

Magic Angle Spinning ◽

Atomic Resolution ◽

Magic Angle ◽

Nmr Structure Determination ◽

Structure Information ◽

Resolution Structure

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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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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Faculty Opinions recommendation of Atomic-resolution protein structure determination by cryo-EM.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738869969.793582672 ◽

2021 ◽

Author(s):

Edward Yu

Keyword(s):

Protein Structure ◽

Structure Determination ◽

Protein Structure Determination ◽

Atomic Resolution

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Initial phases from phosphate SAD in the direct methods determination of RNA

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314093875 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C612-C612

Author(s):

Blaine Mooers ◽

Tzanko Doukov ◽

Tina McKay ◽

Akila Venkataramany ◽

Victoria Mooers

Keyword(s):

Diffraction Data ◽

Structure Determination ◽

Hybrid Approach ◽

Direct Methods ◽

Atomic Resolution ◽

Anomalous Diffraction ◽

Long Wavelength ◽

Minimum Number ◽

Wavelength Radiation ◽

Phase Angles

Atomic resolution diffraction data from crystals of double-stranded RNAs can often resist automated structure determination by ab initio methods including charge flipping and traditional direct methods. Often it is possible to obtain quick success at direct methods structure determination by supplying the positions of one or more heavier atoms, which are used to calculate a starting set of phase angles. Long wavelength radiation such as that near the iron K absorption edge can be used to measure the weak anomalous diffraction data from phosphorous atoms in the RNA backbones. These anomalous diffraction data can be used to locate the positions of some of the phosphorous atoms. Next, the phosphorous positions can be used to provide initial phases for direct methods structure determination with atomic resolution diffraction data collected with shorter wavelength radiation. We tested this hybrid approach with two double-stranded RNAs, one with 31 unique phosphates and a second with 44 unique phosphates. We used a combination of programs including those in the CCP4, SHELX, and Sir program suites. We varied the number of sweeps of images collected at the iron edge to find the minimum number (and hence minimum exposure) required to find enough of the phosphate substructure for success at direct methods with the native data before collecting atomic resolution diffraction data with the short wavelength radiation. Our results suggest that diffraction data could be collected at these two wavelengths from a single crystal to avoid problems with non-isomorphism.

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