Using a partial atomic model from medium-resolution cryo-EM to solve a large crystal structure

Medium-resolution cryo-electron microscopy maps, in particular when they include a significant number of α-helices, may allow the building of partial models that are useful for molecular-replacement searches in large crystallographic structures when the structures of homologs are not available and experimental phasing has failed. Here, as an example, the solution of the structure of a bacteriophage portal using a partial 30% model built into a 7.8 Å resolution cryo-EM map is shown. Inspection of the self-rotation function allowed the correct oligomerization state to be determined, and density-modification procedures using rotation matrices and a mask based on the cryo-EM structure were critical for solving the structure. A workflow is described that may be applicable to similar cases and this strategy is compared with direct use of the cryo-EM map for molecular replacement.

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Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

Crystals ◽

10.3390/cryst11030273 ◽

2021 ◽

Vol 11 (3) ◽

pp. 273

Author(s):

Yoshita Srivastava ◽

Rachel Bonn-Breach ◽

Sai Shashank Chavali ◽

Geoffrey M. Lippa ◽

Jermaine L. Jenkins ◽

...

Keyword(s):

Rna Recognition Motif ◽

Rna Recognition ◽

Molecular Replacement ◽

Hydrophobic Core ◽

Anomalous Diffraction ◽

Recognition Motif ◽

Determination Process ◽

Experimental Phasing ◽

Crystal Contacts ◽

Apical Loop

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

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Cryo-EM structure of the human cohesin-NIPBL-DNA complex

Science ◽

10.1126/science.abb0981 ◽

2020 ◽

Vol 368 (6498) ◽

pp. 1454-1459 ◽

Cited By ~ 20

Author(s):

Zhubing Shi ◽

Haishan Gao ◽

Xiao-chen Bai ◽

Hongtao Yu

Keyword(s):

Electron Microscopy ◽

Sister Chromatid ◽

Base Pair ◽

Adenosine Triphosphatase ◽

Cryo Electron Microscopy ◽

Synergistic Activation ◽

Medium Resolution ◽

Chromatid Cohesion ◽

Dna Complex ◽

Insight Into

As a ring-shaped adenosine triphosphatase (ATPase) machine, cohesin organizes the eukaryotic genome by extruding DNA loops and mediates sister chromatid cohesion by topologically entrapping DNA. How cohesin executes these fundamental DNA transactions is not understood. Using cryo–electron microscopy (cryo-EM), we determined the structure of human cohesin bound to its loader NIPBL and DNA at medium resolution. Cohesin and NIPBL interact extensively and together form a central tunnel to entrap a 72–base pair DNA. NIPBL and DNA promote the engagement of cohesin’s ATPase head domains and ATP binding. The hinge domains of cohesin adopt an “open washer” conformation and dock onto the STAG1 subunit. Our structure explains the synergistic activation of cohesin by NIPBL and DNA and provides insight into DNA entrapment by cohesin.

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Cylindrical Similarity Measurement for Helices in Medium-Resolution Cryo-Electron Microscopy Density Maps

Journal of Chemical Information and Modeling ◽

10.1021/acs.jcim.0c00010 ◽

2020 ◽

Vol 60 (5) ◽

pp. 2644-2650 ◽

Cited By ~ 2

Author(s):

Salim Sazzed ◽

Peter Scheible ◽

Maytha Alshammari ◽

Willy Wriggers ◽

Jing He

Keyword(s):

Electron Microscopy ◽

Similarity Measurement ◽

Cryo Electron Microscopy ◽

Density Maps ◽

Medium Resolution

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Using Phaser and ensembles to improve the performance of SIMBAD

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319015031 ◽

2020 ◽

Vol 76 (1) ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Adam J. Simpkin ◽

Felix Simkovic ◽

Jens M. H. Thomas ◽

Martin Savko ◽

Andrey Lebedev ◽

...

Keyword(s):

Model Identification ◽

Structural Similarity ◽

Test Cases ◽

Target Sequence ◽

Molecular Replacement ◽

Search Models ◽

Termination Criteria ◽

Alternative Approach ◽

The Relationship ◽

Rotation Function

The conventional approach to search-model identification in molecular replacement (MR) is to screen a database of known structures using the target sequence. However, this strategy is not always effective, for example when the relationship between sequence and structural similarity fails or when the crystal contents are not those expected. An alternative approach is to identify suitable search models directly from the experimental data. SIMBAD is a sequence-independent MR pipeline that uses either a crystal lattice search or MR functions to directly locate suitable search models from databases. The previous version of SIMBAD used the fast AMoRe rotation-function search. Here, a new version of SIMBAD which makes use of Phaser and its likelihood scoring to improve the sensitivity of the pipeline is presented. It is shown that the additional compute time potentially required by the more sophisticated scoring is counterbalanced by the greater sensitivity, allowing more cases to trigger early-termination criteria, rather than running to completion. Using Phaser solved 17 out of 25 test cases in comparison to the ten solved with AMoRe, and it is shown that use of ensemble search models produces additional performance benefits.

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On the application of the expected log-likelihood gain to decision making in molecular replacement

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318004357 ◽

2018 ◽

Vol 74 (4) ◽

pp. 245-255 ◽

Cited By ~ 13

Author(s):

Robert D. Oeffner ◽

Pavel V. Afonine ◽

Claudia Millán ◽

Massimo Sammito ◽

Isabel Usón ◽

...

Keyword(s):

Decision Making ◽

Measurement Errors ◽

Single Atom ◽

Molecular Replacement ◽

Acta Cryst ◽

Quickest Path ◽

Experimental Phasing ◽

Log Likelihood ◽

Replacement Solution ◽

Search Order

Molecular-replacement phasing of macromolecular crystal structures is often fast, but if a molecular-replacement solution is not immediately obtained the crystallographer must judge whether to pursue molecular replacement or to attempt experimental phasing as the quickest path to structure solution. The introduction of the expected log-likelihood gain [eLLG; McCoyet al.(2017),Proc. Natl Acad. Sci. USA,114, 3637–3641] has given the crystallographer a powerful new tool to aid in making this decision. The eLLG is the log-likelihood gain on intensity [LLGI; Read & McCoy (2016),Acta Cryst.D72, 375–387] expected from a correctly placed model. It is calculated as a sum over the reflections of a function dependent on the fraction of the scattering for which the model accounts, the estimated model coordinate error and the measurement errors in the data. It is shown how the eLLG may be used to answer the question `can I solve my structure by molecular replacement?'. However, this is only the most obvious of the applications of the eLLG. It is also discussed how the eLLG may be used to determine the search order and minimal data requirements for obtaining a molecular-replacement solution using a given model, and for decision making in fragment-based molecular replacement, single-atom molecular replacement and likelihood-guided model pruning.

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Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21004052 ◽

2021 ◽

Vol 77 (5) ◽

pp. 156-162

Author(s):

Jia Q. Truong ◽

Stephanie Nguyen ◽

John B. Bruning ◽

Keith E. Shearwin

Keyword(s):

Heavy Atom ◽

Protein Structures ◽

Molecular Replacement ◽

Hen Egg White Lysozyme ◽

Phase Problem ◽

Egg White Lysozyme ◽

Mad Phasing ◽

Experimental Phasing ◽

Anomalous Signal ◽

Hen Egg White

The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

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Macromolecular crystal data phased by negative-stained electron-microscopy reconstructions

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444910002763 ◽

2010 ◽

Vol 66 (5) ◽

pp. 514-521 ◽

Cited By ~ 10

Author(s):

Stefano Trapani ◽

Guy Schoehn ◽

Jorge Navaza ◽

Chantal Abergel

Keyword(s):

Electron Microscopy ◽

Streptomyces Coelicolor ◽

Medium Size ◽

Molecular Replacement ◽

X Ray Diffraction ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

Large Particles ◽

One Step ◽

Type Ii Dehydroquinase

The combination of transmission electron microscopy with X-ray diffraction data is usually limited to relatively large particles. Here, the approach is continued one step further by utilizing negative staining, a technique that is of wider applicability than cryo-electron microscopy, to produce models of medium-size proteins suitable for molecular replacement. The technique was used to solve the crystal structure of the dodecameric type II dehydroquinase enzyme fromCandida albicans(∼190 kDa) and that of the orthologousStreptomyces coelicolorprotein.

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Structural and Mutational Analysis of tRNA Intron-Splicing Endonuclease from Thermoplasma acidophilum DSM 1728: Catalytic Mechanism of tRNA Intron-Splicing Endonucleases

Journal of Bacteriology ◽

10.1128/jb.00713-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8339-8346 ◽

Cited By ~ 10

Author(s):

Young Kwan Kim ◽

Kenji Mizutani ◽

Kyung-Hee Rhee ◽

Ki-Hyun Nam ◽

Won Ho Lee ◽

...

Keyword(s):

Catalytic Mechanism ◽

Mutational Analysis ◽

Main Role ◽

Molecular Replacement ◽

Intron Splicing ◽

Thermoplasma Acidophilum ◽

Measuring Mechanism ◽

Crystallographic Structures ◽

Trna Substrate

ABSTRACT In archaea, RNA endonucleases that act specifically on RNA with bulge-helix-bulge motifs play the main role in the recognition and excision of introns, while the eukaryal enzymes use a measuring mechanism to determine the positions of the universally positioned splice sites relative to the conserved domain of pre-tRNA. Two crystallographic structures of tRNA intron-splicing endonuclease from Thermoplasma acidophilum DSM 1728 (EndA Ta ) have been solved to 2.5-Å and 2.7-Å resolution by molecular replacement, using the 2.7-Å resolution data as the initial model and the single-wavelength anomalous-dispersion phasing method using selenomethionine as anomalous signals, respectively. The models show that EndA Ta is a homodimer and that it has overall folding similar to that of other archaeal tRNA endonucleases. From structural and mutational analyses of H236A, Y229F, and K265I in vitro, we have demonstrated that they play critical roles in recognizing the splice site and in cleaving the pre-tRNA substrate.

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