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Several SER Structures, Strategies, Surfaces, and Such. The Derewenda Lab University of Virginia Earth Day, 2008. Sponsored by the letter S. Protein crystallized in our group by the surface engineering approach, with solved crystal structures (as of March 2008) .

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several ser structures strategies surfaces and such

Several SER Structures, Strategies, Surfaces, and Such.

The Derewenda Lab

University of Virginia

Earth Day, 2008.

Sponsored by the letter S.

Protein crystallized in our group by the surface engineering approach, with solved crystal structures (as of March 2008)
  • The RGSL domain of PDZRhoGEF

(Longenecker KL, et al. & Derewenda ZS. Structure, 2001, 9:559-69)

  • The LcrV antigen of the plague-causing bacterium Yersinia pestis

(Derewenda, U. et al. & Waugh, D.S. Structure, 2001, 9:559-69)

  • Product of the YkoFB. subtilis gene

(Devedjiev, Y. et al. & Derewenda, Z.S. J Mol Biol. 2004, 343:395-406)

  • Product of the YdeNB. subtilis gene

(Janda, I. et al. & Derewenda, Z.S. Acta Crystallogr 2004, D60: 1101-1107)

  • Product of the Hsp33B. subtilis gene

(Janda, I. et al. & Derewenda, Z.S. Structure 2004, 12:1901-1907)

  • The product of the YkuDB. subtilis gene

(Bielnicki, J. et al. & Derewenda, Z.S. Proteins, 2006, 62:144-51)

  • The Ohr protein of B. subtilis

(Cooper, D. et al. & Derewenda, Z.S. Acta Cryst 2007, D63:1269-1273)

  • The N-DCX domain of human doublecortin

(Cierpicki, et al. & Derewenda, Z.S. Proteins; 2006:D64:874-882)

  • The p23-like domain of the human nuclear migration NudC protein

(Zheng, M. et al. & Derewenda, Z.S. in preparation)

  • APC 1446 Bacillus subtilis

(Derewenda, U. et al. & Derewenda, Z.S. in preparation)

  • DinB Bacillus subtilis

(Cooper, D.R. et al. & Derewenda, Z.S. in preparation)

  • Tm0439 – VanR family transcription factor

(Zheng, M. et al. & Derewenda, Z.S. in preparation)

  • TM1865 – endonuclease V

(Utepbergenov, D. et al. & Derewenda, Z.S. in preparation)

  • Tm0260 – Phosphate transport regulator

(Zheng, M. et al. & Derewenda, Z.S. in preparation)

  • Tm1382 – NUDIX hydrolase (Possible mutT family member)

(Choi, W.C., et al. & Derewenda, Z.S. in preparation)


Publications by other groups reporting crystallization of novel proteins(green),or preparations of higher quality crystal forms(red)of proteins previously crystallized, by the SER method (as of March 2008)

The CUE:ubiquitin complex

(Prag G et al., & Hurley JH, Cell. 2003, 113:609-20)

Unactivated insulin-like growth factor-1 receptor kinase

(Munshi, S. et al. & Kuo, L.C. Acta Cryst. 2003, D59:1725-1730)

Human choline acetyltransferase

(Kim, A-R., et al. & Shilton, B. H. Acta Cryst. 2005, D61, 1306-1310)

Activated factor XI in complex with benzamidine

(Jin, L., et al. & Strickler, J.E. Acta Cryst. 2005, D61, 1418-1425)

Axon guidance protein MICAL

(Nadella, M., et al. & Amzel, M.L. PNAS, 2005, 102, 16830-16835)

Functionally intact Hsc70 chaperone

(Jiang, J., et al. & Sousa, R. Molecular Cell, 2005, 20, 513-524)

EscJ protein from the Type III secretion system

(Yip, C.K., et al. & Strynadka, N.C.J. Nature, 435: 702-707)

L-rhamnulose kinase from E. coli

(Grueninger D, & Schultz, G.E.) J. Mol. Biol, 2006, 359, 787-797)

T4 vertex gp24 protein

(Boeshans, K.M.., et al. & Ahvazi, B. Protein Expr. Purif., 2006, 49, 235-243.

Borrelia burgdorferi outer surface protein A

(Makabe, K., et al. & Koide, S. Protein Science., 2006, 15, 1907-1914)

SH2 domain from the SH2-B murine adapter protein

(Hu, J., & Hubbard, S.R J. Mol. Biol., 2006, 361, 69-79)

Mycoplasma arthriditis-derived mitogen

(Guo, Y., et al., & Li, H. J., Acta Cryst. 2006, F62, 238-241)

KChIP1 – Kv4.3 T1 complex

(Pioletti, M., et al. & Minor, D. L., Nature, Str & Mol Bio. 2006, 13: 988-995

Kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP-PNP (R126A)

(Zhao, B., et al & Schackenberg, C.G., Protein Science, 2007, 16, 2761-2769)

Human IL-7 bound to unglycosylated and glycosylated forms of its  receptor

(Wickham, J. Jr. and Walsh, S.T.R., Acta Crystallographica, 2007, F63, 865-869)

Human cyclin B1 (C167S, C283S, C350S, E183A, E184A)

(Petri, E.T., et al. & Basavappa, R. Cell Cycle, 2007, 6: 1342-1349)

Candida boidinii formate dehydrogenase

(Schirwitz, K., Schmidt, A. & Lamzin, V.S. Protein Science, 2007, 16: 1146-1156)

EpsI/EpsJ complex

(Yanez, M.E., et al., Hol, W.G.J. J. Mol. Biol., 2008, 375:471-486)

Periplasmic domain of E. coli YidC

(Paetzel, M & Oliver, D.C. J. Biol. Chem., 2008, 283:5208-5216)

Candida boidinii formate dehydrogenase

(Schirwitz, K., Schmidt, A. & Lamzin, V.S. Protein Science, 2007, 16: 1146-1156)

-ketoacyl acyl carrier protein from Streptococcus pneumoniae (FabF)

(Parthasarathy, G. et al., & Soisson, Stephen, M. 2008, Acta Crystallographica, D64:141-148)

our current ser strategy
Our Current SER strategy
  • Target evaluation and selection—

See the slides after the acknowledgements for information on:

      • PSI Structural Genomics Knowledgebase

      • DisMeta (a disorder meta-server)
      • XtalPred
  • Expression of Wild Type – taken through to crystallization trials.
    • Performed on a chromatography system and eluted as a gradient to determine optimal washing concentration of imidazole.
    • We will work with WT crystals for ~2 months before undertaking mutagenesis.
  • Mutation Site and Replacement Residue selection
    • We use the SERp server and use the three best sites.
    • We make Ala and Tyr variants for the top 3 clusters.
  • QuikChange mutatgenesis
    • We make them all at once.
  • Purification, crystallization.
    • We use gravity columns and wash with the imidazole concentration determined for the wild type protein. Some lab members like to purify all 6 at once, others like to purify the 1A and 1Y variants first.

Site 1) K49, E50, E51

Site 2) K173, E174

Site 3) K25, K26, K28


Endonuclease V (TM1865), is a DNA repair enzyme. It cleaves a second phosphodiester bond (in 5’ direction) from a deaminated base.

Recognizes an unusually broad range of irregularities in the DNA structure:

hairpins, unpaired/mispaired bases, deaminated residues, abasic sites etc



  • Found throughout nature – homologs in human, bacteria, archaea
  • Structure unknown, function is believed to be DNA repair
  • However, E. coli deficient in EndoV are generally normal and resistant to mutagens (except nitrosating agents). The enzyme is important for the resistance of E.coli to mutagenesis during nitrate/nitrite respiration.
  • Enzyme is used for mutagenesis and for high throughput detection of mutations in clinical samples
  • E. coli enzyme commercially available from NEB
  • Thermatoga enzyme commercially available from Fermentas
tm1865 crystallization structure solution
TM1865 – crystallization, structure solution
  • Purifies and crystallizes easily as a wild type, no need to apply SER
  • Crystals of SeMet derivative were obtained directly from the JCSG screen, (24% PEG1500, 20% glycerol ) using 1.5 M NaCl in reservoir.
  • P212121, a=69.27, b=71.37, c=119.78
  • Scaled at 2.7Å
  • 3 molecules per ASU, solution from Shelx, model with Solve/Resolve and O.
  • Current R-factor 18% (Rfree – 29%) further refinement is still necessary
tm1865 overall structure
TM1865 – overall structure

Asymmetric trimer Monomer

tm1865 belongs to the rnasehi superfamily
TM1865 belongs to the RNaseHI superfamily.

RNaseHI overall structure: Structure of catalytic center:

Catalytic site consists of 3-5 residues coordinating two metal ions (Mg or Mn).

Metals are known to be crucial for catalysis: one is believed to lower the pKa of attacking nucleophile (water), another is believed to stabilize the negative charge on the formed pentacovalent intermediate.

rnasehi fold family proteins in pdb with rnasehi like fold
RNaseHI fold family – proteins in PDB with RNaseHI-like fold
  • RNaseHI - cleaves RNA strand if it is in duplex with DNA
  • UvrC – major part of bacterial DNA repair system. Recognizes irregularities in the DNA structure
  • RuvC – Holliday junction resolvase
  • Retroviral Integrase – integrates viral genome into host’s DNA
  • Argonaute – Important players in RNA interference
  • Transposase – incorporates DNA fragments into another DNA
  • Mitochondrial Resolvase
  • RNaseHII - cleaves RNA strand if it is in duplex with DNA

All these proteins cleave DNA or RNA strands to perform their function

closest homologs in pdb
Closest homologs in PDB

2nrt (magenta) subdomain of UvrC protein from 2dqe – protein with unknown function

TM. Uvr is a major DNA repair system in bacteria UPF0125 proteins are found in some

organisms living in extreme conditions

tm 1865 conclusions
Tm 1865 Conclusions
  • Endonuclease V belongs to RNase H superfamily of proteins
  • There are no structures of Endonuclease V in PDB but 2 recent structures have similar fold; there are more similar structures known within RNAse H superfamily.
  • Catalytic sites of UvrC and EndonucleaseV are identical

Site 1) E188,K119,K122

Site 2) K2, K3

Site 3) E30, K31


Unrooted tree of the proteins of the GntR family

HTH motif

Effector binding domain

Four subfamilies: FadR, HutC, MocR, and YtrA.

FadR subfamily: FadR and VanR

FadR 1st, regroups 40%

All helical C-terminal domain

7 or 6 helices

VanR-like regulators, 170 aa and 150 aa

Regulation of oxidized substrates

Rigali, S. et al. J. Biol. Chem. 2002;277:12507-12515




crystal contact of tm0439
Crystal contact of Tm0439







Wild type: crystals, poor

Mutant: 130E131K134K2AAA, 1A, good quality

Crystal contact

dna binding domain of tm0439
DNA-Binding domain of Tm0439



















An HTH motif: 2 and 3, tight turn

Superimpose: conserved 2nd structure element, HTH motif: Tm0439: V46-E70, 2HS5: E54-D78, 1E2X: A33-D58

1-2 loops, equal length, conformation

stereo model of tm0439 dna complex
Stereo model of Tm0439-DNA complex











The proposed Tm0439-DNA binding mode

Putative DNA contacts: 4 distinct regions

1: At the N-terminus, side chains of V18, L19, V21, and M13-E17 couldn’t be seen

2: At the beginning of 2 helix, V46 and R47

3: 3, major groove, residues S56, F57, T58, P59 and R61

4: At the tip of the 1-2 hairpin, P78 and R79

effector binding domain of tm0439
Effector-binding domain of Tm0439












C-terminal domain: 6 -helices (4-9) with short connecting loops, form a bundle

1E2X has 7 helices

2HS5 has 6 helices

All helices bundle, superimposed together

the putative switch mechanism of tm0439
The putative switch mechanism of Tm0439






















FadR dimer


Tm0439 dimer


Site 1) K158,E159,K160

Site 2) K77,Q78,E80

Site 3) E47, E49

nudix hydrolase superfamily
Nudix Hydrolase Superfamily
  • Pyrophosphohydrolases that act upon Nucleoside DIphosphate connected to another moiety (X)

Such substrates include (d)NTPs (both canonical and oxidised derivatives), nucleotide sugars and alcohols, dinucleoside polyphosphates (NpnN), dinucleotide coenzymes and capped RNAs.

  • The substrate diversity requires equally diverse chemistries.
  • Tm1382 is classified as a MutT hydrolase by the JCSG, but it is 50% larger than most members of the family.
  • Consensus Nudix Sequence


  • Tm1382 Sequence
    • Gx4Ex5LxREx2EExDV

Current Working Model

some parts are missing
Some parts are missing








************ **********. ****************




*. ****************************** . *****


working-chA EGWSKISAAVLAG---GGG 165

working-chB EGWSKISAAVLL------- 161



some distant homologues top dali hits
Some Distant Homologues(Top Dali Hits)





Found on the PSI Knowledgebase



Site 1) K159,E160

Site 2) K78,E79

Site 3) K100, K101


We thought there was no viable MR model (see below), but thank to the PSI Structural Genomics Knowledgebase, we have the structure.



35% Identity

RFZ=7.3 TFZ=8.8 PAK=0 LLG=74 LLG=74

the surface problem
The Surface problem
  • “In accordance with the assumption that solvent exposure of a residue is directly related to its probability of forming random contacts, accessible surface area might be used as the basis of a reference state to compute the number of random contacts expected.”(Dasgupta1997)‏
  • surface = sum over all atoms.
  • 85% residues have ASA > 0




selection is futile
Selection is futile
  • Area-based comparisons are almost as bad as number based.
  • No ASA or rASA threshold will fix different distributions



patch analysis of crystal contacts
Patch analysis of crystal contacts
  • Jones&Thornton introduced a patch methodology to analyse properties of biologically relevant interfaces on the protein surface.
  • The major problems are:
    • defining a single contact (interface):
      • coordination number (only binary)‏
      • clustering (artifacts)‏
    • sampling the surface:
      • make random interfaces
spherical protein approximation
Spherical protein approximation
  • coordinate system and distance measure:



in 3D:- three (0,2π) angles.- one for each axis.- + r the radius

Pros:- easy to cluster!- with r, mahalanobis



do we need r?

space is the place sun ra
Space is the place ”Sun Ra”
  • We need to measure the distance between atoms to make continuous patches on the surface:
    • the coordinate space affects sampling frequency possibly introducing bias.
  • getting information from pdb files
  • robust ... workflow based ... scalable
  • object oriented
  • outsourcing:
    • Areaimol, Ncont/Act, Stride, MSMS
    • numpy/scipy (k-means clustering)‏
    • scipy-cluster (hierarchical clustering)‏
    • Bio.KDTree (NN distance look-up)‏
    • scikits.ANN (NN k look-up)‏
    • CGAL, CGAL-python (voronoi)‏
    • PyTables (bindings for hdf5)‏
the noble 8 fold path
The noble 8-fold path:

from zenpdb import *

file_name = 'some_pdb_file'

parser = PDBParser(forgive =1)‏


structure = p.get_structure(file_name[0:4])‏

ACTAtomContacts(in_file, structure)‏

residues = einput(structure, 'R')‏

r_x = residues._select_children({}, 'gt', \

'CNT_ACT_X', xtra=True).values()‏

HierarchicalResidueClusters(r_x, dmethod ='mahalanobis', lmethod='average', criterion ='maxclust', t=6)‏

BeQu('new_pdb_file.pdb', structure, 'R', 'H_CLUST')‏

structures around the corner need phasing power
Structures Around the Corner(need phasing power)
  • Tm0260
    • Several data sets diffracting to ~2.2 Å (R32)
    • Should have 8 Seleniums in the ASU
    • MR encouraging
  • Tm1024
    • Lots of beautiful crystals
    • Several data sets to ~ 2.4 Å of 1A and 1Y mutants
    • Only 1 Methionine.
      • Creating several L->M mutations
      • Creating the 1M Mutant (K45M, K46M)
tm0260 putative phosphate regulatory protein
Tm0260Putative phosphate regulatory protein

Site 1) K153,E154,K155

Site 2) E10,E11

Site 3) E78,K79

mr encouraging but
MR encouraging, but…

The closest model is only 16% identical and is symmetrical. Long helices can be seen, but there are no side chain features and the ends are ambiguous.



Zygmunt Derewenda

Jakub Bielnicki

Marvin Cieslik

WonChan Choi

David Cooper

Ulla Derewenda

Monika Kijanska

Natalya Olekhnovich

Darkhan Utepbergenov

Jennifer Wingard

Meiying Zheng

Tomek Boczek

Kasia Grelewska

Gosia Pinkowska

Michal Zawadzki

Eliza Zylkiewic

Los Alamos Nat’l Lab

Tom Terwilliger

Chang Yub Kim


David Eisenberg

Luki Goldschmidt

Tom Holton

Lawrence Berkeley Nat’l Lab

Li-Wei Hung

Minmin Yu (Big Thanks)

Jeff Habel

And ALL ISFI members!

The ISFI is funded by NIH U54 GM074946.

Several slides follow.

dismeta a nesg metaserver http www nmr cabm rutgers edu bioinformatics disorder
DisMeta – a NESG MetaServer

Queries up to 12 different disorder prediction servers.

http kb psi structuralgenomics org kb1

Click Here

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