Development of a universal nanobody-binding Fab module for fiducial-assisted cryo-EM studies of membrane proteins

With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon CDR grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryo-EM structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of ~50 kDa. Using an additional anti-Fab nanobody further facillitated reliable initial 3D structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, humanized, and can be conveniently expressed in E. coli in large amounts, it may not only be useful for structural biology, but also for biomedical applications.

Magnetic Particles used in a New Approach for Designed Protein Crystallization

After more than one hundred and thirty thousand protein structures determined by X-ray crystallography, the challenge of protein crystallization for 3D structure determination remains. In the quest for additives for...

Atomically resolved 3D structural reconstruction of small Quantum Dots

The semiconducting Quantum Dot (QD) has potential applications in light-emitting diodes, single-photon sources and quantum computing due to shape-dependent (opto) electronic properties. Atomic resolution 3D-structure determination is important in understanding...

Accurate inference of the full base-pairing structure of RNA by deep mutational scanning and covariation-induced deviation of activity

Despite the large number of noncoding RNAs in human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained only from costly and time-consuming 3D structure determination. Here, we performed deep mutational scanning of self-cleaving CPEB3 ribozyme by error-prone PCR and showed that a library of <5 × 104 single-to-triple mutants is sufficient to infer 25 of 26 base pairs including non-nested, nonhelical, and noncanonical base pairs with both sensitivity and precision at 96%. Such accurate inference was further confirmed by a twister ribozyme at 100% precision with only noncanonical base pairs as false negatives. The performance was resulted from analyzing covariation-induced deviation of activity by utilizing both functional and nonfunctional variants for unsupervised classification, followed by Monte Carlo (MC) simulated annealing with mutation-derived scores. Highly accurate inference can also be obtained by combining MC with evolution/direct coupling analysis, R-scape or epistasis analysis. The results highlight the usefulness of deep mutational scanning for high-accuracy structural inference of self-cleaving ribozymes with implications for other structured RNAs that permit high-throughput functional selections.

Ribosolve: Rapid determination of three-dimensional RNA-only structures

The discovery and design of biologically important RNA molecules is dramatically outpacing three-dimensional structural characterization. To address this challenge, we present Ribosolve, a hybrid method integrating moderate-resolution cryo-EM maps, chemical mapping, and Rosetta computational modeling, and demonstrate its application to thirteen previously unknown 119-to 338-nucleotide protein-free RNA-only structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length V. cholerae and F. nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and computer-designed spinach-TTR-3, eterna3D-JR_1, and ATP-TTR-3 with and without AMP. Blind challenges, prospective compensatory mutagenesis, internal controls, and simulation benchmarks validate the Ribosolve models and establish that modeling convergence is quantitatively predictive of model accuracy. These results demonstrate that RNA-only 3D structure determination can be rapid and routine.

Accurate inference of the full base-pairing structure of RNA by deep mutational scanning and covariation-induced deviation of activity

ABSTRACTDespite the transcription of noncoding RNAs in 75% of the human genome and their roles in many diseases include cancer, we know very little about them due to lack of structural clues. The centerpiece of the structural clues is the full RNA base-pairing structure of secondary and tertiary contacts that can be precisely obtained only from costly and inefficient 3D structure determination. Here, we performed deep mutational scanning of self-cleaving CPEB3 ribozyme by error-prone PCR and showed that a library of <5×104 single-to-triple mutants is sufficient to infer all 26 including nonhelical and noncanonical base pairs at the precision of a single false positive. The accurate inference, further confirmed by a twister ribozyme, is resulted from covariation analysis by utilizing both functional and nonfunctional variants for unsupervised learning, followed by restrained optimization. The result highlights the usefulness of deep mutational scanning for high-accuracy structural inference.