Bioisis

"RPA DNA-Binding Core with 20mer"

Experimental SAS Curve

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Low_res_thumbnail

Experimental Mass

83,600 Da

Experimental Details for BID:  DBC20Y
Experiment ID: 111
Collected at: ALS BL 12.3.1
Contributors: Brosey, CA ,  Yan, C ,  Tsutakawa, SE ,  Heller, WT ,  Rambo, RP ,  Tainer, JA ,  Ivanov, I ,  Chazin, WJ
SAXS profile of the DNA-binding core of heterotrimeric Replication Protein A (RPA70ABC/32D/14) in complex with a 20-nucleotide substrate (dCCACCCCCCCCCCCCCCCCC). The construct under study contains RPA domains 70A, 70B, and 70C from the RPA70 subunit, domain 32D from the RPA32 subunit, and the single-domain subunit RPA14. DNA-binding domains include 70A, 70B, 70C, and 32D.
Data was collected as a concentration series (1X, 2X, 4X) from 0.29 mL gel filtration fractions collected from a Superdex 200 HR 10/30 gel filtration column. The reported profile is from the 4X sample with a concentration of 10-11 mg/mL.

Electron Pair Distribution

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       Dmax → 114 Å


Guinier Plot

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     Guinier Rg → 34.9 Å

Real Space Rg → 35.11 Å

The Guinier plot is used to estimate the radius of gyration, Rg, which is taken from the slope of a line observed at low scattering angles (typically in the range where q* Rg < 1.3). This should be in reasonable agreement with the real space Rg.


Kratky Plot

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The Kratky plot can be used to visually assess the degree of "unfoldedness" of a protein or RNA sample. The plot of a well-behaved folded protein approaches the baseline at high q values creating a parabolic shape.


GASBOR Model

GASBOR result was determined with the following:

Space GroupP1
Chi6.7
NSD | RMSD1.2 
variance(NSD | RMSD)0.12 
Number of Models in Average14 
Superimposed model14 

Additional Experimental Details
Title

RPA DNA-Binding Core with 20mer

Description

SAXS profile of the DNA-binding core of heterotrimeric Replication Protein A (RPA70ABC/32D/14) in complex with a 20-nucleotide substrate (dCCACCCCCCCCCCCCCCCCC). The construct under study contains RPA domains 70A, 70B, and 70C from the RPA70 subunit, domain 32D from the RPA32 subunit, and the single-domain subunit RPA14. DNA-binding domains include 70A, 70B, 70C, and 32D.

Publication

A new structural framework for integrating replication protein A into DNA processing machinery, Nucleic Acids Research, PMID: 23303776

Contributors

Brosey, CA ,  Yan, C ,  Tsutakawa, SE ,  Heller, WT ,  Rambo, RP ,  Tainer, JA ,  Ivanov, I ,  Chazin, WJ

Genomics and Proteomics

The experiment is composed of 4 genes/ORFs

Abbreviated name: 70ABC

Annotation: RPA70ABC (RPA70 residues 181-616) MW: 49.4 kDa, contains 2 additional N-terminal residues (GP) from removal of 6X-His fusion tag.

GPQSKVVPIA SLTPYQSKWT ICARVTNKSQ IRTWSNSRGE GKLFSLELVD ESGEIRATAF NEQVDKFFPL IEVNKVYYFS KGTLKIANKQ FTAVKNDYEM TFNNETSVMP CEDDHHLPTV QFDFTGIDDL ENKSKDSLVD IIGICKSYED ATKITVRSNN REVAKRNIYL MDTSGKVVTA TLWGEDADKF DGSRQPVLAI KGARVSDFGG RSLSVLSSST IIANPDIPEA YKLRGWFDAE GQALDGVSIS DLKSGGVGGS NTNWKTLYEV KSENLGQGDK PDYFSSVATV VYLRKENCMY QACPTQDCNK KVIDQQNGLY RCEKCDTEFP NFKYRMILSV NIADFQENQW VTCFQESAEA ILGQNAAYLG ELKDKNEQAF EE
categoryamino acid composition(%)
HydrophobicI(5.5) V(7.1) L(6.5) M(1.3) A(6.3) G(6.5) P(3.1)
AromaticF(4.7) W(1.6) Y(3.7)
HydrophilicR(3.7) K(7.9) E(7.1) D(7.1) Q(4.7) N(6.3) H(0.5) S(8.1) T(6.0) C(2.4)

Abbreviated name: 32D

Annotation: RPA32D (RPA32 residues 43-171) MW: 14.6 kDa

MAQHIVPCTI SQLLSATLVD EVFRIGNVEI SQVTIVGIIR HAEKAPTNIV YKIDDMTAAP MDVRQWVDTD DTSSENTVVP PETYVKVAGH LRSFQNKKSL VAFKIMPLED MNEFTTHILE VINAHMVLSK
categoryamino acid composition(%)
HydrophobicI(9.2) V(12.3) L(6.2) M(4.6) A(6.9) G(2.3) P(4.6)
AromaticF(3.1) W(0.8) Y(1.5)
HydrophilicR(3.1) K(5.4) E(6.2) D(6.2) Q(3.8) N(4.6) H(3.8) S(6.2) T(8.5) C(0.8)

Abbreviated name: RPA14

Annotation: RPA14, MW: 13.6 kDa, contains 2 additional N-terminal residues (GP) from removal of 6X-His fusion tag.

GPMVDMMDLP RSRINAGMLA QFIDKPVCFV GRLEKIHPTG KMFILSDGEG KNGTIELMEP LDEEISGIVE VVGRVTAKAT ILCTSYVQFK EDSHPFDLGL YNEAVKIIHD FPQFYPLGIV QHD
categoryamino acid composition(%)
HydrophobicI(8.9) V(8.1) L(8.1) M(4.9) A(4.1) G(8.9) P(6.5)
AromaticF(5.7) W(0.0) Y(2.4)
HydrophilicR(3.3) K(5.7) E(7.3) D(7.3) Q(3.3) N(2.4) H(3.3) S(4.1) T(4.1) C(1.6)

Abbreviated name: D20

Annotation: ssDNA substrate in complex

CCACCCCCCC CCCCCCCCCC
categoryResidue composition (%)
Nucleotides G (0.0) C (95.0) A (5.0) U (0.0) T (0.0)
Mass: 6,047 Da | Total residues: 20