Iterative screen optimization maximizes the efficiency of macromolecular crystallization

Jones, H.G.; Wrapp, Daniel; Gilman, M.S.A.; Battles, Michael B.; Wang, Nianshuang; Sacerdote, Sofia; Chuang, Gwo Yu; Kwong, Peter D.; McLellan, Jason S.

doi:10.1107/s2053230x18017338

Cited by 8 publications

(7 citation statements)

References 32 publications

(33 reference statements)

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“…In the "down" conformation, the RBDs were tightly packed against 31 the top of the S2 subunit, preventing binding by ACE2 (Gui et al, 2017). Subsequent 32 experiments have corroborated this phenomenon and similar dynamics have been observed in 33 MERS-CoV S, SARS-CoV-2 S and in alphacoronavirus S proteins (Jones et al, 2019;34 Kirchdoerfer et al, 2018;Pallesen et al, 2017;Walls, 2020;Wrapp et al, 2020;Yuan et al, 35 2017). Due to the relatively low abundance of particles that can be observed by cryo-EM with 36 three RBDs in the up conformation, it is thought that this conformation may correspond to an 37 energetically unstable state (Kirchdoerfer et al, 2018;Pallesen et al, 2017).…”

Section: Introductionmentioning

confidence: 83%

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

Wrapp

Vlieger

Corbett

et al. 2020

Preprint

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ABSTRACTThe pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

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Section: Introductionmentioning

confidence: 83%

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

Wrapp

Vlieger

Corbett

et al. 2020

Preprint

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“…At the same time, novel crystalline cocktails could be discovered, including their distinct crystal morphologies. As an efficient tool, ISO was extremely suitable for crystallizing protein samples that required a narrow range of concentrations of precipitants …”

Section: New Progress In Crystallization Condition Screening Practicementioning

confidence: 99%

An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening

Qin

Xie

Zhang

et al. 2019

Crystal Research and Technology

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Obtaining diffraction-quality crystals is still a bottleneck for biomacromolecular structure determination using X-ray diffraction. In practice, the bottleneck mainly comprises two aspects: difficulties in screening and in optimization, and both of them need screening kits. The paper aims at an investigation of the chemical components employed in 86 common commercial screening kits, and comparison with those in the Biological Macromolecules Crystallization Database (BMCD). First, the 86 screening kits are systematically analyzed in terms of design methods, classification, applicable scope, and manufacturing company. Second, the chemical components applied in these screening kits are investigated in detail in terms of the classification and utilization frequency, which are compared with those in BMCD. Third, four main chemical reagents of screening kits including salts, buffers, precipitants, and additives are analyzed in detail. The top ten utilized chemical components are listed. Last, the paper summarizes new progress and further innovation in crystallization condition screening practice and screening kits design. The analysis result would be a valuable reference for rational selection and design of screening kits, and even for personalized screening of a target biomacromolecule, which would provide new ideas to solve the bottleneck problem of biomacromolecular structure determination.

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“…Meanwhile, some commercial devices have been developed to satisfy the requirement from academic centers and pharmaceutical companies. The fully automatic crystallization imaging system, Rockimager1500, was designed by the Formulatrix [11] company. However, it was only able to analyze the experimental drop and not crystals in the drop.…”

Section: Introductionmentioning

confidence: 99%

Protein Crystal Instance Segmentation Based on Mask R-CNN

et al. 2021

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Protein crystallization is the bottleneck in macromolecular crystallography, and crystal recognition is a very important step in the experiment. To improve the recognition accuracy by image classification algorithms further, the Mask R-CNN model is introduced for the detection of protein crystals in this paper. Because the protein crystal image is greatly affected by backlight and precipitate, the contrast limit adaptive histogram equalization (CLAHE) is applied with Mask R-CNN. Meanwhile, the Transfer Learning method is used to optimize the parameters in Mask R-CNN. Through the comparison experiments between this combined algorithm and the original algorithm, it shows that the improved algorithm can effectively improve the accuracy of segmentation.

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Iterative screen optimization maximizes the efficiency of macromolecular crystallization

Cited by 8 publications

References 32 publications

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-domain Camelid Antibodies

An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening

Protein Crystal Instance Segmentation Based on Mask R-CNN

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